Cellular distribution of glucose and monocarboxylate transporters in human brain white matter and multiple sclerosis lesions.

To ensure efficient energy supply to the high demanding brain, nutrients are transported into brain cells via specific glucose (GLUT) and monocarboxylate transporters (MCT). Mitochondrial dysfunction and altered glucose metabolism are thought to play an important role in the progression of neurodegenerative diseases, including multiple sclerosis (MS). Here, we investigated the cellular localization of key GLUT and MCT proteins in human brain tissue of non-neurological controls and MS patients. We show that in control brain tissue GLUT and MCT proteins were abundantly expressed in a variety of central nervous system cells, particularly in microglia and endothelial cells. In active MS lesions, GLUTs and MCTs were highly expressed in infiltrating leukocytes and reactive astrocytes. Astrocytes manifest increased MCT1 staining and maintain GLUT expression in inactive lesions, whereas demyelinated axons exhibit significantly reduced GLUT3 and MCT2 immunoreactivity in inactive lesions. Finally, we demonstrated that the co-transcription factor peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α), an important protein involved in energy metabolism, is highly expressed in reactive astrocytes in active MS lesions. Overexpression of PGC-1α in astrocyte-like cells resulted in increased production of several GLUT and MCT proteins. In conclusion, we provide for the first time a comprehensive overview of key nutrient transporters in white matter brain samples. Moreover, our data demonstrate an altered expression of these nutrient transporters in MS brain tissue, including a marked reduction of axonal GLUT3 and MCT2 expression in chronic lesions, which may impede efficient nutrient supply to the hypoxic demyelinated axons thereby contributing to the ongoing neurodegeneration in MS. GLIA 2014;62:1125-1141.

Activation of a HIF1alpha-PPARgamma axis underlies the integration of glycolytic and lipid anabolic pathways in pathologic cardiac hypertrophy.

Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1alpha and PPARgamma, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1alpha activates glycolytic genes and PPARgamma, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1alpha in mice prevents hypertrophy-induced PPARgamma activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1alpha-PPARgamma axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.

Comparative assessment of intimal hyperplasia development after 14 days in two different experimental settings: tissue culture versus ex vivo continuous perfusion of human saphenous vein.

BACKGROUND: Intimal hyperplasia (IH) is a vascular remodeling process which often leads to failure of arterial bypass or hemodialysis access. Experimental and clinical work have provided insight in IH development; however, further studies under precise controlled conditions are required to improve therapeutic strategies to inhibit IH development. Ex vivo perfusion of human vessel segments under standardized hemodynamic conditions may provide an adequate experimental approach for this purpose. Therefore, chronically perfused venous segments were studied and compared to traditional static culture procedures with regard to functional and histomorphologic characteristics as well as gene expression. MATERIALS AND METHODS: Static vein culture allowing high tissue viability was performed as previously described. Ex vivo vein support system (EVVSS) was performed using a vein support system consisting of an incubator with a perfusion chamber and a pump. EVVSS allows vessel perfusion under continuous flow while maintaining controlled hemodynamic conditions. Each human saphenous vein was divided in two parts, one cultured in a Pyrex dish and the other part perfused in EVVSS for 14days. Testing of vasomotion, histomorphometry, expression of CD 31, Factor VIII, MIB 1, alpha-actin, and PAI-l were determined before and after 14days of either experimental conditions. RESULTS: Human venous segments cultured under traditional or perfused conditions exhibited similar IH after 14 days as shown by histomorphometry. Smooth-muscle cell (SMC) was preserved after chronic perfusion. Although integrity of both endothelial and smooth-muscle cells appears to be maintained in both culture conditions as confirmed by CD31, factor VIII, and alpha-actin expression, a few smooth-muscle cells in the media stained positive for factor VIII. Cell-proliferation marker MIB-1 was also detected in the two settings and PAI-1 mRNA expression and activity increased significantly after 14 days of culture and perfusion. CONCLUSION: This study demonstrates the feasibility to chronically perfuse human vessels under sterile conditions with preservation of cellular integrity and vascular contractility. To gain insights into the mechanisms leading to IH, it will now be possible to study vascular remodeling not only under static conditions but also in hemodynamic environment mimicking as closely as possible the flow conditions encountered in reconstructive vascular surgery.

NLRP3 inflammasome is required in murine asthma in the absence of aluminum adjuvant.

Background: Inflammasome activation with the production of IL-1 beta received substantial attention recently in inflammatory diseases. However, the role of inflammasome in the pathogenesis of asthma is not clear. Using an adjuvant-free model of allergic lung inflammation induced by ovalbumin (OVA), we investigated the role of NLRP3 inflammasome and related it to IL-1R1 signaling pathway.Methods: Allergic lung inflammation induced by OVA was evaluated in vivo in mice deficient in NLRP3 inflammasome, IL-1R1, IL-1 beta or IL-1 alpha. Eosinophil recruitment, Th2 cytokine, and chemokine levels were determined in bronchoalveolar lavage fluid, lung homogenates, and mediastinal lymph node cells ex vivo.Results: Allergic airway inflammation depends on NLRP3 inflammasome activation. Dendritic cell recruitment into lymph nodes, Th2 lymphocyte activation in the lung and secretion of Th2 cytokines and chemokines are reduced in the absence of NLRP3. Absence of NLRP3 and IL-1 beta is associated with reduced expression of other proinflammatory cytokines such as IL-5, IL-13, IL-33, and thymic stromal lymphopoietin. Furthermore, the critical role of IL-1R1 signaling in allergic inflammation is confirmed in IL-1R1-, IL-1 beta-, and IL-1 alpha-deficient mice.Conclusion: NLRP3 inflammasome activation leading to IL-1 production is critical for the induction of a Th2 inflammatory allergic response.

Na(+)-K(+)-ATPase gene expression during in vitro development of rat fetal forebrain.

The existence of at least three isoforms of Na(+)-K(+)-ATPase in adult brain tissues [alpha 1, kidney type; alpha 2 [or alpha(+)]; alpha 3] suggests that these genes might be regulated in a cell-specific and time-dependent manner during development. We have studied this question in serum-free aggregating cell cultures of mechanically dissociated rat fetal telencephalon. At the protein level, the relative rate of synthesis of the pool of alpha 1-, alpha 2-, and alpha 3-subunits increased approximately twofold over 15 days of culture, leading to a marked increase in the immunochemical pool of alpha-subunits as measured by a panspecific polyclonal antibody. Concomitantly, Na(+)-K(+)-ATPase enzyme-specific activity increased three- (lower forebrain) to sixfold (upper forebrain). The transcripts of all three alpha-isoforms and beta-subunit were detected in vitro in similar proportion to the level observed in vivo. alpha 3-mRNA (3.7 kb) was more abundant than alpha 1 (3.7 kb) or alpha 2 (5.3 and 3.4 kb). Cytosine arabinoside (0.4 microM) and cholera toxin (0.1 microM) were used to selectively eliminate glial cells or neurons, respectively. It was found that alpha 2-mRNA is predominantly transcribed in glial cell cultures, whereas alpha 3- and beta 1-mRNA (2.7, 2.3, and 1.8 kb) are predominant in neuronal cultures.

Vitamin D deficiency and a CYP27B1-1260 promoter polymorphism are associated with chronic hepatitis C and poor response to interferon-alfa based therapy.

BACKGROUND & AIMS: Vitamin D is an important immune modulator and preliminary data indicated an association between vitamin D deficiency and sustained virologic response (SVR) rates in hepatitis C virus (HCV) genotype 1 patients. We, therefore, performed a comprehensive analysis on the impact of vitamin D serum levels and of genetic polymorphisms with functional relevance within the vitamin D cascade on chronic hepatitis C and its treatment. METHODS: Vitamin D serum levels, genetic polymorphisms within the vitamin D receptor and 1α-hydroxylase were determined in a cohort of 468 HCV genotype 1, 2, and 3 infected patients who were treated with interferon-alfa based regimens. RESULTS: Chronic hepatitis C was associated with a high incidence of severe vitamin D deficiency compared to controls (25(OH)D(3)<10 ng/ml in 25% versus 12%, p<0.00001). 25(OH)D(3) deficiency correlated with SVR in HCV genotype 2 and 3 patients (50% and 81% SVR for patients with and without severe vitamin D deficiency, respectively, p<0.0001). In addition, the CYP27B1-1260 promoter polymorphism rs10877012 had substantial impact on 1,25-dihydroxyvitamin D serum levels (72, 61, and 60 pmol/ml for rs10877012 AA, AC, and CC, respectively, p=0.04) and on SVR rates in HCV genotype 1, 2, and 3 infected patients (77% and 65% versus 42% for rs10877012 AA, AC, and CC, respectively, p=0.02). CONCLUSIONS: Chronic hepatitis C virus infection is associated with vitamin D deficiency. Reduced 25-hydroxyvitamin D levels and CYP27B1-1260 promoter polymorphism leading to reduced 1,25-dihydroxyvitamin D levels are associated with failure to achieve SVR in HCV genotype 1, 2, and 3 infected patients.

Mitomycin C with continuous fluorouracil or with cisplatin in combination with radiotherapy for locally advanced anal cancer (European Organisation for Research and Treatment of Cancer phase II study 22011-40014).

PURPOSE: To assess the feasibility and activity of radio-chemotherapy with mitomycin C (MMC) and cisplatin (CDDP) in locally advanced squamous cell anal carcinoma with reference to radiotherapy (RT) combined with MMC and fluorouracil (5-FU). PATIENTS AND METHODS: Patients with measurable disease >4 cmN0 or N+ received RT (36Gy+2 week gap+23.4Gy) with either MMC/CDDP or MMC/5-FU (MMC 10mg/m(2) d1 of each sequence; 5-FU 200mg/m(2)/day c.i.v. daily; CDDP 25mg/m(2) weekly). Forty patients/arm were needed to exclude a RECIST objective response rate (ORR), 8 weeks after treatment, of <75% (Fleming 1, alpha=10%, beta=10%). RESULTS: The ORR was 79.5% (31/39) (lower bound confidence interval [CI]: 68.8%) with MMC/5-FU versus 91.9% (34/ 37) (lower bound CI: 82.8%) with MMC/CDDP. In the MMC/5-FU group, two patients (5.1%) discontinued treatment due to toxicity versus 11 (29.7%) in the MMC/CDDP group. Nine grade 3 haematological events occurred with MMC/CDDP versus none with 5-FU/MMC. The rate of other toxicities did not differ. There was no toxic death. Thirty-one patients in the MMC/5-FU arm (79.5%) and 18 in the MMC/CDDP arm (48.6%) were fully compliant with the protocol treatment (p=0.005). CONCLUSIONS: Radio-chemotherapy with MMC/CDDP seems promising as only MMC/CDDP demonstrated enough activity (RECIST ORR >75%) to be tested further in phase III trials; MMC/5-FU did not. MMC/CDDP also had an overall acceptable toxicity profile.

Reduction of ARNT in myeloid cells causes immune suppression and delayed wound healing.

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor that binds to partners to mediate responses to environmental signals. To investigate its role in the innate immune system, floxed ARNT mice were bred with lysozyme M-Cre recombinase animals to generate lysozyme M-ARNT (LAR) mice with reduced ARNT expression. Myeloid cells of LAR mice had altered mRNA expression and delayed wound healing. Interestingly, when the animals were rendered diabetic, the difference in wound healing between the LAR mice and their littermate controls was no longer present, suggesting that decreased myeloid cell ARNT function may be an important factor in impaired wound healing in diabetes. Deferoxamine (DFO) improves wound healing by increasing hypoxia-inducible factors, which require ARNT for function. DFO was not effective in wounds of LAR mice, again suggesting that myeloid cells are important for normal wound healing and for the full benefit of DFO. These findings suggest that myeloid ARNT is important for immune function and wound healing. Increasing ARNT and, more specifically, myeloid ARNT may be a therapeutic strategy to improve wound healing.

Myelin basic protein content of aggregating rat brain cell cultures treated with cytokines and/or demyelinating antibody: effects of macrophage enrichment.

The demyelinative potential of the cytokines interleukin-1 alpha (IL-1 alpha), interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) has been investigated in myelinating aggregate brain cell cultures. Treatment of myelinated cultures with these cytokines resulted in a reduction in myelin basic protein (MBP) content. This effect was additively increased by anti-myelin/oligodendrocyte glycoprotein (alpha-MOG) in the presence of complement. Qualitative immunocytochemistry demonstrated that peritoneal macrophages, added to the fetal telencephalon cell suspensions at the start of the culture period, successfully integrated into aggregate cultures. Supplementing the macrophage component of the cultures in this fashion resulted in increased accumulation of MBP. The effect of IFN-gamma on MBP content of cultures was not affected by the presence of macrophages in increased numbers.

Vascular Integrins: Therapeutic and Imaging Targets of Tumor Angiogenesis

Cells, including endothelial cells, continuously sense their surrounding environment and rapidly adapt to changes in order to assure tissues and organs homeostasis. The extracellular matrix (ECM) provides a physical scaffold for cell positioning and represents an instructive interface allowing cells to communicate over short distances. Cell surface receptors of the integrin family emerged through evolution as essential mediators and integrators of ECM-dependent communication. In preclinical studies, pharmacological inhibition of vascular integrins suppressed angiogenesis and inhibited tumor progression. alpha(V)beta(3) and alpha(V)beta(5) were the first integrins targeted to suppress tumor angiogenesis. Subsequently, additional integrins, in particular alpha(1)beta(1), alpha(2)beta(1), alpha(5)beta(1), and alpha(6)beta(4), emerged as potential therapeutic targets. Integrin inhibitors are currently tested in clinical trials for their safety and antiangiogenic/antitumor activity. In this chapter, we review the role of integrins in angiogenesis and present recent advances in the use of integrin antagonists as potential therapeutics in cancer and discuss future perspectives.

Apparent versus true gene expression changes of three hypoxia-related genes in autopsy derived tissue and the importance of normalisation.

The aim of our work was to show how a chosen normal-isation strategy can affect the outcome of quantitative gene expression studies. As an example, we analysed the expression of three genes known to be upregulated under hypoxic conditions: HIF1A, VEGF and SLC2A1 (GLUT1). Raw RT-qPCR data were normalised using two different strategies: a straightforward normalisation against a single reference gene, GAPDH, using the 2(-ΔΔCt) algorithm and a more complex normalisation against a normalisation factor calculated from the quantitative raw data from four previously validated reference genes. We found that the two different normalisation strategies revealed contradicting results: normalising against a validated set of reference genes revealed an upregulation of the three genes of interest in three post-mortem tissue samples (cardiac muscle, skeletal muscle and brain) under hypoxic conditions. Interestingly, we found a statistically significant difference in the relative transcript abundance of VEGF in cardiac muscle between donors who died of asphyxia versus donors who died from cardiac death. Normalisation against GAPDH alone revealed no upregulation but, in some instances, a downregulation of the genes of interest. To further analyse this discrepancy, the stability of all reference genes used were reassessed and the very low expression stability of GAPDH was found to originate from the co-regulation of this gene under hypoxic conditions. We concluded that GAPDH is not a suitable reference gene for the quantitative analysis of gene expression in hypoxia and that validation of reference genes is a crucial step for generating biologically meaningful data.

PVHL is a regulator of glucose metabolism and insulin secretion in pancreatic beta cells.

Insulin secretion from pancreatic beta cells is stimulated by glucose metabolism. However, the relative importance of metabolizing glucose via mitochondrial oxidative phosphorylation versus glycolysis for insulin secretion remains unclear. von Hippel-Lindau (VHL) tumor suppressor protein, pVHL, negatively regulates hypoxia-inducible factor HIF1alpha, a transcription factor implicated in promoting a glycolytic form of metabolism. Here we report a central role for the pVHL-HIF1alpha pathway in the control of beta-cell glucose utilization, insulin secretion, and glucose homeostasis. Conditional inactivation of Vhlh in beta cells promoted a diversion of glucose away from mitochondria into lactate production, causing cells to produce high levels of glycolytically derived ATP and to secrete elevated levels of insulin at low glucose concentrations. Vhlh-deficient mice exhibited diminished glucose-stimulated changes in cytoplasmic Ca(2+) concentration, electrical activity, and insulin secretion, which culminate in impaired systemic glucose tolerance. Importantly, combined deletion of Vhlh and Hif1alpha rescued these phenotypes, implying that they are the result of HIF1alpha activation. Together, these results identify pVHL and HIF1alpha as key regulators of insulin secretion from pancreatic beta cells. They further suggest that changes in the metabolic strategy of glucose metabolism in beta cells have profound effects on whole-body glucose homeostasis.

Cannabidiol Protects against Doxorubicin-Induced Cardiomyopathy by Modulating Mitochondrial Function and Biogenesis.

Doxorubicin (DOX) is a widely used, potent chemotherapeutic agent; however, its clinical application is limited because of its dose-dependent cardiotoxicity. DOX's cardiotoxicity involves increased oxidative/nitrative stress, impaired mitochondrial function in cardiomyocytes/endothelial cells and cell death. Cannabidiol (CBD) is a nonpsychotropic constituent of marijuana, which is well tolerated in humans, with antioxidant, antiinflammatory and recently discovered antitumor properties. We aimed to explore the effects of CBD in a well-established mouse model of DOX-induced cardiomyopathy. DOX-induced cardiomyopathy was characterized by increased myocardial injury (elevated serum creatine kinase and lactate dehydrogenase levels), myocardial oxidative and nitrative stress (decreased total glutathione content and glutathione peroxidase 1 activity, increased lipid peroxidation, 3-nitrotyrosine formation and expression of inducible nitric oxide synthase mRNA), myocardial cell death (apoptotic and poly[ADP]-ribose polymerase 1 [PARP]-dependent) and cardiac dysfunction (decline in ejection fraction and left ventricular fractional shortening). DOX also impaired myocardial mitochondrial biogenesis (decreased mitochondrial copy number, mRNA expression of peroxisome proliferator-activated receptor γ coactivator 1-alpha, peroxisome proliferator-activated receptor alpha, estrogen-related receptor alpha), reduced mitochondrial function (attenuated complex I and II activities) and decreased myocardial expression of uncoupling protein 2 and 3 and medium-chain acyl-CoA dehydrogenase mRNA. Treatment with CBD markedly improved DOX-induced cardiac dysfunction, oxidative/nitrative stress and cell death. CBD also enhanced the DOX-induced impaired cardiac mitochondrial function and biogenesis. These data suggest that CBD may represent a novel cardioprotective strategy against DOX-induced cardiotoxicity, and the above-described effects on mitochondrial function and biogenesis may contribute to its beneficial properties described in numerous other models of tissue injury.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.