Magnetic Resonance Imaging of Gliomas

Open Access.This work was supported in part by grants CTQ2010-20960-C02-02 to P.L.L. and grant SAF2008-01327 to S.C. A.M.M. held an Erasmus Fellowship from Coimbra University and E.C.C. a predoctoral CSIC contract.Peer Reviewe

In vivo MR approaches to validate the capacity of a new vanadium compound as a promising anti-diabetic drug

Trabajo presentado al 19th Joint Annual Meeting & Exhibition of International Society for Magnetic Resonance in Medicine, celebrado en Montreal (Canada) del 7 al 13 de Mayo de 2011.Peer Reviewe

In vivo HIF-mediated reductive carboxylation is regulated by citrate levels and sensitizes VHL-deficient cells to glutamine deprivation

PMCID: PMC4003458; NIHMSID: NIHMS449661.-- et al.Hypoxic and VHL-deficient cells use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate. To gain insights into the role of HIF and the molecular mechanisms underlying RC, we took advantage of a panel of disease-associated VHL mutants and showed that HIF expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. HIF expression drastically reduced intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate or knocking down PDK-1 and ACLY restored citrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action to maintain lipogenesis. Using [ 1-13C]glutamine, we demonstrated in vivo RC activity in VHL-deficient tumors growing as xenografts in mice. Lastly, HIF rendered VHL-deficient cells sensitive to glutamine deprivation in vitro, and systemic administration of glutaminase inhibitors suppressed the growth of RCC cells as mice xenografts. © 2013 Elsevier Inc.This work was supported by NIH R01 CA122591, the MGH Proton Beam Federal Share Project, and an Astra-Zeneca Award (all to O.I.); funding from the Foundation for Science and Technology (FCT), Portugal (to P.A.G.); MICINN CTQ-2010-20960-CO2-02 (to P.L-L.); and NIHR01 DK075850 (to G.S.).Peer Reviewe

VO(dmpp)2 reverts pre-diabetic features in fatty Zucker rats: MRI/MRS techniques as non-invasive powerful tools in drug development

Trabajo presentado al 29th Annual Scientific Meeting of European Society for Magnetic Resonance In Medicine and Biology, celebrado en Lisboa (Portugal) del 4 la 6 de Octubre de 2012.Peer Reviewe

VO(dmpp)2 normalizes pre-diabetic parameters as assessed by in vivo magnetic resonance imaging and spectroscopy

Type 2 diabetes mellitus has been associated with obesity, metabolic syndrome, cardiovascular diseases and cancer. Attempts have been made for early diagnosis and finding effective drugs to prevent severe consequences and ameliorate the symptoms of this disorder. In this work, the pharmacological properties of VO(dmpp) 2, [bis(1,2-dimethyl-3-hydroxy-4-pyridinonato) oxovanadium(IV)], were in vivo evaluated. For 4 weeks fatty Zucker rats were subjected to a daily dose of VO(dmpp) 2 (44 μmol/kg) and their metabolic profile was followed by assessing different biological parameters at established time points: body weight, subcutaneous fat width and hepatic triglyceride content determined by magnetic resonance imaging and spectroscopy, respectively. A glucose tolerance test was performed at the end of the experiment. After treatment, treated obese rats presented a weight significantly lower than the non-treated obese animals (359.0 ± 11.1 vs. 433.5 ± 6.2 g, P < 0.05), a thinner subcutaneous fat width, and a statistically significant decrease in hepatic triglyceride content (5.41 ± 0.59 vs. 21.03 ± 1.40%, P < 0.0005). Additionally, the glucose intolerant profile of fatty Zucker rats was completely reversed in treated animals (102.3 ± 2.1 vs. 172.4 ± 1.3 mg/100 mL; P < 0.0005). These results reinforce the therapeutic action of VO(dmpp) 2 which shows particular effects on lipid metabolism. © 2012 Elsevier Inc. All rights reserved.This work was supported by the grant CTQ2010-20960-C02-02 from Ministerio de Ciencia e Innovación, Spain, to Pilar López-Larrubia.Peer Reviewe

Metabolic effects of VO(dmpp)2 ¿ an ex vivo1H-HRMAS NMR study to unveil its pharmacological properties

The pharmacological action of the V(IV) compound VO(dmpp)2 was previously evaluated in vivo using obese pre-diabetic Zucker rats treated with this compound for four weeks. Besides the promising results regarding biological parameters indicative of insulin-mimetism, a specific biological activity in mitigating impaired lipid metabolism was observed. This work aims at complementing and reinforcing data formerly attained and it represents an attempt to unveil the effect of this compound at the molecular level, particularly on the metabolic profile of different organs. Ex vivo experiments with tissue samples of the brain, liver and skeletal muscle from treated and non-treated obese Zucker rats, using lean Zucker rats as a control, were carried out by using 1H-HRMAS. The results obtained showed that the lipid liver content, a characteristic of obese rats, significantly decreased after 4 weeks of VO(dmpp)2 treatment, as demonstrated by the quantification of the respective signals in the 1H-HRMAS NMR spectra. In the other analyzed tissues, the differences were not statistically significant, but a trend of a decrease of the abnormal metabolite content in treated obese rats was observed. Importantly, the therapeutic dose used showed no renal toxicity. VO(dmpp)2 was demonstrated to be able to revert the impaired lipid metabolism in vivo and 1H-HRMAS NMR was revealed to be a good tool to simultaneously assess the effects of a drug on the metabolic profile of different organs and tissues.This work was supported by grants from the Ministry of Economy, Industry and Competitivity (SAF2017-83043-R), and by the Program MULTITARGET&VIEW-CM from Community of Madrid, Spain (S-BIO-0170-2006), involving contributions from FEDER and FSE. A. M. M. and M. M. C. A. C. acknowledge Fundação para a Ciência e Tecnologia (FCT), Portugal, for the financial support to the CQC research unit through the project 007630 UID/QUI/00313/2013, co-funded by COMPETE2020-EU

Hypoxia onducible factor (HIF) promotes reductive carboxylation in vivo and renders VHL-deficient cells sensitive to glutamine deprivation

Resumen del póster presentado al Keystone Symposium on PI 3-Kinase and Interplay with Other Signaling Pathways celebrado en Keystone-Colorado (US) del 24 de febrero al 1 de marzo de 2013.Hypoxia is a hallmark of tumor microenvironment. We showed before taht exposure of cells to hypoxia reprograms cell metabolism and directs and IDH1-mediated reductive carboxylation (RC) of glutamine-derived alpha-ketoglutarate (a-KG) for lipogenesis. In addition, Von Hippel-Lindau (VHL)-deficient renal cell carcinoma (RCC) cells use glutamine to generate citrate and lipids through RC of a-KG. to gain insights into the role of HIF and the molecular mechanisms underlying RC we took advantage of a panel of disease-associated VHL mutants and showed that HIF expression is necessary and sufficient for the induction of RC in human RCC cells. Expression of a VHL-independent HIF mutant in VHL-restored RCC cells is sufficient to promote RC. HIF expression drastically reduces intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate, or knocking-down PDK-1 and ACLY enzymes restored intracellularcitrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action, to maintain lipogenesis. Expression of HIF renders hypoxic and VHL-deficient cells addicted to glutamine in vitro. Systemic administration of glutaminase inhibitor (BPTES) suppressed the growth of VHL-deficient human RCC cell lines as xenografts in nude mice. Lastly, we investigated whether RC occurs in vivo. We metabolically labeled mice bearing VHL-deficient tumors by infusing them with 13C-1Glutamine for up to 6 hours and we detected the early formation of labeled citrate in the tumors; thus whe showed for the first time RC in vivo. Our data provide mechanistic insights into the signaling events that mediate hypoxia-induced RC, strongly suggest that RC is an in vivo phenomenon in growing tumors and highlight potential novel therapeutic approaches for treatment of hypoxic and VHL-deficient tumors based on this metabolic signature.Peer reviewe

Loss of vhl in the zebrafish pronephros recapitulates early stages of human clear cell renal cell carcinoma

Patients with von Hippel–Lindau (VHL) disease harbor a germline mutation in the VHL gene leading to the development of several tumor types including clear cell renal cell carcinoma (ccRCC). In addition, the VHL gene is inactivated in over 90% of sporadic ccRCC cases. ‘Clear cell’ tumors contain large, proliferating cells with ‘clear cytoplasm’, and a reduced number of cilia. VHL inactivation leads to the stabilization of hypoxia inducible factors 1a and 2a [HIF1a and HIF2a (HIF2a is also known as EPAS1)] with consequent up-regulation of specific target genes involved in cell proliferation, angiogenesis and erythropoiesis. A zebrafish model with a homozygous inactivation in the VHL gene (vhl−/−) recapitulates several aspects of the human disease, including development of highly vascular lesions in the brain and the retina and erythrocytosis. Here, we characterize for the first time the epithelial abnormalities present in the kidney of the vhl−/− zebrafish larvae as a first step in building a model of ccRCC in zebrafish. Our data show that the vhl−/− zebrafish kidney is characterized by an increased tubule diameter, disorganized cilia, the dramatic formation of cytoplasmic lipid vesicles, glycogen accumulation, aberrant cell proliferation and abnormal apoptosis. This phenotype of the vhl−/− pronephros is reminiscent of clear cell histology, indicating that the vhl−/− mutant zebrafish might serve as a model of early stage RCC. Treatment of vhl−/− zebrafish embryos with a small-molecule HIF2a inhibitor rescued the pronephric abnormalities, underscoring the value of the zebrafish model in drug discovery for treatment of VHL disease and ccRCC

In Vivo HIF-Mediated Reductive Carboxylation Is Regulated by Citrate Levels and Sensitizes VHL-Deficient Cells to Glutamine Deprivation

Hypoxic and VHL-deficient cells use glutamine to generate citrate and lipids through reductive carboxylation (RC) of α-ketoglutarate. To gain insights into the role of HIF and the molecular mechanisms underlying RC, we took advantage of a panel of disease-associated VHL mutants and showed that HIF expression is necessary and sufficient for the induction of RC in human renal cell carcinoma (RCC) cells. HIF expression drastically reduced intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate or knocking down PDK-1 and ACLY restored citrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action to maintain lipogenesis. Using [[superscript 1–13]C]glutamine, we demonstrated in vivo RC activity in VHL-deficient tumors growing as xenografts in mice. Lastly, HIF rendered VHL-deficient cells sensitive to glutamine deprivation in vitro, and systemic administration of glutaminase inhibitors suppressed the growth of RCC cells as mice xenografts.Fundacao para a Ciencia e a TecnologiaNational Institutes of Health (U.S.) (R01 DK075850

Gene-edited healthy donor CAR T cells show superior anti-tumour activity compared to CAR T cells derived from patients with lymphoma in an in vivo model of high-grade lymphoma

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