[Show abstract][Hide abstract] ABSTRACT: Angiogenesis is a central regulator for white (WAT) and brown adipose tissue (BAT) adaptation in the course of obesity. Here
we show that deletion of hypoxia-inducible factor 2α (HIF2α) in adipocytes (by using Fabp4-Cre transgenic mice), but not in
myeloid or endothelial cells, negatively impacted on WAT angiogenesis and promoted WAT inflammation, WAT dysfunction, hepatosteatosis
and systemic insulin resistance in obesity. Importantly, adipocyte HIF2α regulated vascular endothelial growth factor (VEGF)
expression and angiogenesis of the obese BAT as well as its thermogenic function. Consistently, obese adipocyte-specific HIF2α-deficient
mice displayed BAT dysregulation, associated with reduced levels of uncoupling protein-1 (UCP1) and a dysfunctional thermogenic
response to cold exposure. VEGF administration reversed WAT and BAT inflammation and BAT dysfunction in adipocyte HIF2α-deficient
mice. Together, adipocyte HIF2α is protective against maladaptation to obesity and metabolic dysregulation by promoting angiogenesis
in both WAT and BAT and by counteracting obesity-mediated BAT dysfunction.
Preview · Article · Nov 2015 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: The antiepileptic drug valproic acid (VPA) has been shown to influence the neural differentiation and neurite outgrowth of neural stem cells. Sympathoadrenal progenitor cells share properties with neural stem cells and are considered a potential cell source in the treatment of neurodegenerative diseases. The present study therefore aims at modulating the neural differentiation potential of these cells by treatment with the histone deacetylase inhibitor VPA. We studied the epigenetic effects of VPA in two culture conditions: suspension conditions aimed to expand adrenomedullary sympathoadrenal progenitors within free-floating chromospheres and adherent cell cultures optimized to derive neurons. Treatment of chromospheres with VPA may launch neuronal differentiation mechanisms and improve their neurogenic potential upon transplantation. However, also transplantation of differentiated functional neurons could be beneficial. Treating chromospheres for 7 days with clinically relevant concentrations of VPA (2 mm) revealed a decrease of neural progenitor markers Nestin, Notch2 and Sox10. Furthermore, VPA initiated catecholaminergic neuronal differentiation indicated by upregulation of the neuronal marker β-III-tubulin, the dopaminergic transcription factor Pitx3 and the catecholaminergic enzymes TH and GTPCH. In adherent neural differentiation conditions, VPA treatment improved the differentiation of sympathoadrenal progenitor cells into catecholaminergic neurons with significantly elevated levels of nor- and epinephrine. In conclusion, similar to neural stem cells, VPA launches differentiation mechanisms in sympathoadrenal progenitor cells that result in increased generation of functional neurons. Thus, data from this study will be relevant to the potential use of chromaffin progenitors in transplantation therapies of neurodegenerative diseases.Molecular Psychiatry advance online publication, 24 February 2015; doi:10.1038/mp.2015.3.
No preview · Article · Feb 2015 · Molecular Psychiatry
[Show abstract][Hide abstract] ABSTRACT: Context and objective:
Pheochromocytomas and paragangliomas (PGLs) are neuroendocrine tumors of sympathetic or parasympathetic paraganglia. Nearly 40% of PGLs are caused by germline mutations. The present study investigated the effect of genetic alterations on metabolic networks in PGLs.
Homogenates of 32 sporadic PGLs and 48 PGLs from patients with mutations in SDHB, SDHD, SDHAF-2, VHL, RET, and NF-1 were subjected to proton ((1)H) nuclear magnetic resonance (NMR) spectroscopy at 500 MHz for untargeted and HPLC tandem mass spectrometry for targeted metabolite profiling.
(1)H NMR spectroscopy identified 28 metabolites in PGLs of which 12 showed genotype-specific differences. Part of these results published earlier reported low complex II activity (P < .0001) and low ATP/ADP/AMP content (P < .001) in SDH-related PGLs compared with sporadics and PGLs of other genotypes. Extending these results, low levels of N-acetylaspartic acid (NAA; P < .05) in SDH tumors and creatine (P < .05) in VHL tumors were observed compared with sporadics and other genotypes. Positive correlation was observed between NAA and ATP/ADP/AMP content (P < .001) and NAA and complex II activity (P < .0001) of PGLs. Targeted purine analysis in PGLs showed low adenine in cluster 1 compared with cluster 2 tumors (SDH P < .0001; VHL P < .05) whereas lower levels (P < .05) of guanosine and hypoxanthine were observed in RET tumors compared with SDH tumors. Principal component analysis (PCA) of metabolites could distinguish PGLs of different genotypes.
The present study gives a comprehensive picture of alterations in energy metabolism in SDH- and VHL-related PGLs and establishes the interrelationship of energy metabolism and amino acid and purine metabolism in PGLs.
Full-text · Article · Dec 2014 · Journal of Clinical Endocrinology & Metabolism
[Show abstract][Hide abstract] ABSTRACT: Pheochromocytoma is a rare but potentially lethal neuroendocrine tumor arising from catecholamine producing chromaffin cells. Especially for metastatic pheochromocytoma, the availability of animal models is essential for developing novel therapies. For evaluating therapeutic outcome in rodent pheochromocytoma models reliable quantification of multiple organ lesions depends on dedicated small animal in vivo imaging, which is still challenging and only available at specialized research facilities. Here, we investigated whether whole-body fluorescence imaging and monitoring of urinary free monoamines provide suitable parameters for measuring tumor progression in a murine allograft model of pheochromocytoma. We generated an mCherry-expressing mouse pheochromocytoma cell line by lentiviral gene transfer. These cells were injected subcutaneously into nude mice to perform whole-body fluorescence imaging of tumor development. Urinary free monoamines were measured by liquid chromatography with tandem mass spectrometry. Tumor fluorescence intensity and urinary outputs of monoamines showed tumor growth-dependent increases ( p < .001) over the 30 days of monitoring post tumor engraftment. Concomitantly, systolic blood pressure was increased significantly during tumor growth. Tumor volume correlated significantly ( p < .001) and strongly with tumor fluorescence intensity (r = 0.946) and urinary outputs of dopamine (r = 0.952), methoxytyramine (r = 0.947), norepinephrine (r = 0.756) and normetanephrine (r = 0.949). Dopamine and methoxytyramine outputs allowed for detection of lesions at diameters below 2.3 mm. Our results demonstrate that MPC-mCherry cell tumors are functionally similar to human pheochromocytoma. Both tumor fluorescence intensity and urinary outputs of free monoamines provide precise parameters of tumor progression in this subcutaneous mouse model of pheochromocytoma. This animal model will allow for testing new treatment strategies for chromaffin cell tumors.
[Show abstract][Hide abstract] ABSTRACT: Context: Mutations of succinate dehydrogenase A/B/C/D genes (SDHx) increase susceptibility to development of pheochromocytomas and paragangliomas (PPGLs), with particularly high rates of malignancy associated with SDHB mutations. Objective: We assessed whether altered succinate dehydrogenase product-precursor relationships, manifested by differences in tumor ratios of succinate to fumarate or other metabolites, might aid in identifying and stratifying patients with SDHx mutations. Design, Setting and Patients: PPGL tumor specimens from 233 patients, including 45 with SDHx mutations, were provided from eight tertiary referral centers for mass spectrometric analyses of Krebs cycle metabolites. Main outcome measure: Diagnostic performance of the succinate:fumarate ratio for identification of pathogenic SDHx mutations. Results: SDH-deficient PPGLs were characterized by 25-fold higher succinate and 80% lower fumarate, cis-aconitate and isocitrate tissue levels than PPGLs without SDHx mutations. Receiver-operating characteristic curves for use of ratios of succinate to fumarate or to cis-aconitate and isocitrate to identify SDHx mutations indicated areas under curves of 0.94 to 0.96; an optimal cut-off of 97.7 for the succinate:fumarate ratio provided a diagnostic sensitivity of 93% at a specificity of 97% to identify SDHX-mutated PPGLs. Succinate:fumarate ratios were higher in both SDHB-mutated and metastatic tumors than in those due to SDHD/C mutations or without metastases. Conclusions: Mass spectrometric-based measurements of ratios of succinate:fumarate and other metabolites in PPGLs offer a useful method to identify patients for testing of SDHx mutations, with additional utility to quantitatively assess functionality of mutations and metabolic factors responsible for malignant risk.
[Show abstract][Hide abstract] ABSTRACT: We present a method to efficiently culture primary chromaffin progenitors from the adult bovine adrenal medulla in a defined, serum-free monolayer system. Tissue is dissociated and plated for expansion under support by the mitogen basic fibroblast growth factor (bFGF). The cultures, although not homogenous, contain a subpopulation of cells expressing the neural stem cell marker Hes3 that also propagate. In addition, Hes3 is also expressed in the adult adrenal medulla from where the tissue is taken. Differentiation is induced by bFGF withdrawal and switching to Neurobasal medium containing B27. Following differentiation, Hes3 expression is lost, and cells acquire morphologies and biomarker expression patterns of chromaffin cells and dopaminergic neurons. We tested the effect of different treatments that we previously showed regulate Hes3 expression and cell number in cultures of fetal and adult rodent neural stem cells. Treatment of the cultures with a combination of Delta4, Angiopoietin2, and a Janus kinase inhibitor increases cell number during the expansion phase without significantly affecting catecholamine content levels. Treatment with cholera toxin does not significantly affect cell number but reduces the ratio of epinephrine to norepinephrine content and increases the dopamine content relative to total catecholamines. These data suggest that this defined culture system can be used for target identification in drug discovery programs and that the transcription factor Hes3 may serve as a new biomarker of putative adrenomedullary chromaffin progenitor cells.
Full-text · Article · May 2014 · STEM CELLS TRANSLATIONAL MEDICINE
[Show abstract][Hide abstract] ABSTRACT: Context: About 35% of patients with pheochromocytoma/paraganglioma carry a germ-line mutation in one of the ten main susceptibility genes. The recent introduction of next-generation sequencing will allow the analysis of all these genes in one run. When positive, the analysis is generally unequivocal due to the association between a germ-line mutation and a concordant clinical presentation or positive family history. When genetic analysis reveals a novel mutation with no clinical correlates, particularly in presence of a missense variant, the question arises whether the mutation is pathogenic or a rare polymorphism. Objective: We report a case of a 35 yr old patient operated for a pheochromocytoma who turned out to be carrier of a novel SDHD (succinate-dehydrogenase subunit D) missense mutation. With no positive family history nor clinical correlates, we decided to perform additional analyses to test the mutation clinical significance. Methods: We performed in-silico analysis, tissue LOH analysis, immunohistochemistry, Western blot analysis, SDH enzymatic assay and measurement of the succinate/fumarate concentration ratio in the tumor tissue by tandem mass spectrometry. Results: While the in-silico analysis gave contradictory results according to the different methods, all the other tests demonstrated that the SDH complex was conserved and normally active. We therefore came to the conclusion that the variant was a non-pathogenic polymorphism. Conclusions: Advancements in technology facilitate genetic analysis of patients with pheochromocytoma but also offer new challenges to the clinician who, in some cases, needs clinical correlates and/or functional tests to give significance to the results of the genetic assay.
No preview · Article · Apr 2014 · The Journal of Clinical Endocrinology and Metabolism
[Show abstract][Hide abstract] ABSTRACT: Hypoxia has wide-ranging impact in normal physiology and disease processes. This stimulus evokes changes in gene expression mediated by transcription factors termed hypoxia-inducible factors (HIFs) that affect numerous processes: angiogenesis, cell survival, cellular metabolism, stem cell self-renewal and multipotency, migration, invasiveness, and metastatic progression in tumor cells. Over the past decade, increasing numbers of reports have emerged documenting differential roles of HIF1α and HIF2α in these processes. In cells of the sympathoadrenal lineage, both HIFs differentially mediate influences of hypoxia on catecholamine synthesis and secretion, but HIF2α signaling has particularly prominent functions in regulating developmental processes of growth and differentiation. This chapter discusses the role of HIF2α and HIF1α in the context of the development, phenotypic features, and functions of chromaffin cells. Moreover, current knowledge about tumor formation in cells of the sympathoadrenal lineage, leading to catecholamine-producing pheochromocytomas and paragangliomas, is analyzed in the light of the HIF2α signaling network.
No preview · Article · Sep 2013 · Advances in pharmacology (San Diego, Calif.)
[Show abstract][Hide abstract] ABSTRACT: Purpose:
Pheochromocytomas and paragangliomas (PGL) are neuroendocrine tumors of sympathetic and parasympathetic paraganglia. This study investigated the relationships between genotype-specific differences in mitochondrial function and catecholamine content in PGL tumors.
Respiratory chain enzyme assays and (1)H-nuclear magnetic resonance (NMR) spectroscopy at 500 MHz were conducted on homogenates of 35 sporadic PGLs and 59 PGLs from patients with hereditary mutations in succinate dehydrogenase subunits B and D (SDHB, SDHD), succinate dehydrogenase assembly factor 2, von Hippel-Lindau (VHL), rearranged during transfection (RET), neurofibromatosis type 1 (NF1), and myc-associated factor X.
In SDHx-related PGLs, a significant decrease in complex II activity (P < 0.0001) and a significant increase in complex I, III, and IV enzyme activities were observed when compared to sporadic, RET, and NF1 tumors. Also, a significant increase in citrate synthase (P < 0.0001) enzyme activity was observed in SDHx-related PGLs when compared to sporadic-, VHL-, RET-, and NF1-related tumors. An increase in succinate accumulation (P < 0.001) and decrease in ATP/ADP/AMP accumulation (P < 0.001) was observed when compared to sporadic PGLs and PGLs of other genotypes. Positive correlations (P < 0.01) were observed between respiratory chain complex II activity and total catecholamine content and ATP/ADP/AMP and total catecholamine contents in tumor tissues.
This study for the first time establishes a relationship between determinants of energy metabolism, like activity of respiratory chain enzyme complex II, ATP/ADP/AMP content, and catecholamine content in PGL tumors. Also, this study for the first time successfully uses NMR spectroscopy to detect catecholamines in PGL tumors and provides ex vivo evidence for the accumulation of succinate in PGL tumors with an SDHx mutation.
Full-text · Article · May 2013 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPC) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing.
Full-text · Article · Dec 2012 · Molecular and Cellular Endocrinology
[Show abstract][Hide abstract] ABSTRACT: Here, we describe a novel method utilizing double stable isotope ultra performance liquid chromatography-tandem mass spectrometry to measure tissue contents and activity of phenylethanolamine N-methyltransferase (PNMT), the enzyme responsible for synthesis of the stress hormone, epinephrine. The method is based on measurement of deuterium-labeled epinephrine produced from the reaction of norepinephrine with deuterium-labeled S-adenosyl-L-methionine as the methyl donor. In addition to enzyme activity, the method allows for determination of tissue contents of PNMT using human recombinant enzyme for calibration. The calibration curve for epinephrine was linear over the range of 0.1 to 5,000 pM, with 0.5 pM epinephrine representing the lower limit of quantification. The calibration curve relating PNMT to production of deuterium-labeled epinephrine was also linear from 0.01 to 100 ng PNMT. Intra- and inter-assay coefficients of variation were respectively 12.8 % (n = 10) and 10.9 to 13.6 % (n = 10). We established utility of the method by showing induction of the enzyme by dexamethasone in mouse pheochromocytoma cells and strong relationships to PNMT gene expression and tissue epinephrine levels in human pheochromocytomas. Development of this assay provides new possibilities for investigations focusing on regulation of PNMT, the crucial final enzyme responsible for synthesis of epinephrine, the primary fight-or-flight stress hormone.
Preview · Article · Dec 2012 · Analytical and Bioanalytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: Background:
Measurements of plasma normetanephrine and metanephrine provide a useful diagnostic test for phaeochromocytoma, but this depends on appropriate reference intervals. Upper cut-offs set too high compromise diagnostic sensitivity, whereas set too low, false-positives are a problem. This study aimed to establish optimal reference intervals for plasma normetanephrine and metanephrine.
Blood samples were collected in the supine position from 1226 subjects, aged 5-84 y, including 116 children, 575 normotensive and hypertensive adults and 535 patients in whom phaeochromocytoma was ruled out. Reference intervals were examined according to age and gender. Various models were examined to optimize upper cut-offs according to estimates of diagnostic sensitivity and specificity in a separate validation group of 3888 patients tested for phaeochromocytoma, including 558 with confirmed disease.
Plasma metanephrine, but not normetanephrine, was higher (P < 0.001) in men than in women, but reference intervals did not differ. Age showed a positive relationship (P < 0.0001) with plasma normetanephrine and a weaker relationship (P = 0.021) with metanephrine. Upper cut-offs of reference intervals for normetanephrine increased from 0.47 nmol/L in children to 1.05 nmol/L in subjects over 60 y. A curvilinear model for age-adjusted compared with fixed upper cut-offs for normetanephrine, together with a higher cut-off for metanephrine (0.45 versus 0.32 nmol/L), resulted in a substantial gain in diagnostic specificity from 88.3% to 96.0% with minimal loss in diagnostic sensitivity from 93.9% to 93.6%.
These data establish age-adjusted cut-offs of reference intervals for plasma normetanephrine and optimized cut-offs for metanephrine useful for minimizing false-positive results.
No preview · Article · Oct 2012 · Annals of Clinical Biochemistry
[Show abstract][Hide abstract] ABSTRACT: The differentiation of dopamine-producing neurons from chromaffin progenitors might represent a new valuable source for replacement therapies in Parkinson's disease. However, characterization of their differentiation potential is an important prerequisite for efficient engraftment. Based on our previous studies on isolation and characterization of chromaffin progenitors from adult adrenals, this study investigates their potential to producedopaminergic neurons and means to enhance their dopaminergic differentiation. Chromaffin progenitors grown in sphere culture showed an increased expression of nestin and Mash1 indicating an increase of the progenitor subset. Pro-neurogenic culture conditions induced the differentiation into neurons positive for neural markers β-III-tubulin, MAP2 and TH accompanied by a decrease of Mash1 and nestin. Furthermore, Notch2 expression decreased concomitantly with a down-regulation of downstream effectors Hes1 and Hes5 responsible for self-renewal and proliferation maintenance of progenitor cells. Chromaffin progenitor-derived neurons secreted dopamine upon stimulation by potassium. Strikingly, treatment of differentiating cells with retinoic and ascorbic acid resulted in a twofold increase of dopamine secretion while norepinephrine and epinephrine were decreased. Initiation of dopamine synthesis and neural maturation is controlled by Pitx3 and Nurr1. Both, Pitx3 and Nurr1 were identified in differentiating chromaffin progenitors. Along with the gained dopaminergic function, electrophysiology revealed features of mature neurons such as sodium channels and the capability to fire multiple action potentials. In summary, this study elucidates the capacity of chromaffin progenitor cells to generate functional dopaminergic neurons indicating their potential use in cell replacement therapies.
[Show abstract][Hide abstract] ABSTRACT: Pheochromocytomas (PCC) and paragangliomas (PGL) are genetically heterogeneous neural crest-derived neoplasms. Recently we identified germline mutations in a new tumor suppressor susceptibility gene, MAX (MYC-associated factor X), which predisposes carriers to PCC. How MAX mutations contribute to PCC/PGL and associated phenotypes remain unclear. This study aimed to examine the prevalence and associated phenotypic features of germline and somatic MAX mutations in PCC/PGL. Design: We sequenced MAX in 1,694 patients with PCC or PGL (without mutations in other major susceptibility genes) from 17 independent referral centers. We screened for large deletions/duplications in 1,535 patients using a multiplex PCR-based method. Somatic mutations were searched for in tumors from an additional 245 patients. The frequency and type of MAX mutation was assessed overall and by clinical characteristics.
Sixteen MAX pathogenic mutations were identified in 23 index patients. All had adrenal tumors, including 13 bilateral or multiple PCCs within the same gland (P < 0.001), 15.8% developed additional tumors at thoracoabdominal sites, and 37% had familial antecedents. Age at diagnosis was lower (P = 0.001) in MAX mutation carriers compared with nonmutated cases. Two patients (10.5%) developed metastatic disease. A mutation affecting MAX was found in five tumors, four of them confirmed as somatic (1.65%). MAX tumors were characterized by substantial increases in normetanephrine, associated with normal or minor increases in metanephrine.
Germline mutations in MAX are responsible for 1.12% of PCC/PGL in patients without evidence of other known mutations and should be considered in the genetic work-up of these patients.
Full-text · Article · Mar 2012 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Dehydroepiandrosterone producing adrenocortical zona reticularis and the adrenal medulla are in direct contact and are highly intermingled in many species. This results in potentially strong paracrine influences of high local dehydroepiandrosterone concentrations on the adrenal medulla. Dehydroepiandrosterone has neuroprotective properties and increases neural stem cell proliferation and neurogenesis. Therefore, we aimed to establish its effects on chromaffin progenitor cell proliferation and differentiation. Previously, we successfully isolated chromaffin progenitors from bovine adrenal medulla in spherical cultures, so-called chromospheres. Seven days treatment of chromospheres with dehydroepiandrosterone at high concentrations (100 μM) hampered proliferation of chromaffin progenitors. Under differentiation conditions, dehydroepiandrosterone in the presence of retinoic acid, increased tyrosine hydroxylase and decreased dopamine-β-hydroxylase mRNA expression. In addition, there was a tendency to increase dopamine contents. Dehydroepiandrosterone/retinoic acid is therefore suggested to induce dopaminergic differentiation from chromaffin progenitor cells. Furthermore, the high dehydroepiandrosterone concentrations present in the fetal and adult adrenal may play an important role in adrenomedullary cell proliferation and differentiation.
No preview · Article · Dec 2010 · Molecular and Cellular Endocrinology
[Show abstract][Hide abstract] ABSTRACT: Phaeochromocytomas and paragangliomas (PPGLs) are highly heterogeneous tumours with variable catecholamine biochemical phenotypes and diverse hereditary backgrounds. This analysis of 18 catecholamine-related plasma and urinary biomarkers in 365 patients with PPGLs and 846 subjects without PPGLs examined how catecholamine metabolomic profiles are impacted by hereditary background and relate to variable hormone secretion. Catecholamine secretion was assessed in a subgroup of 156 patients from whom tumour tissue was available for measurements of catecholamine contents. Among all analytes, the free catecholamine O-methylated metabolites measured in plasma showed the largest tumour-related increases relative to the reference group. Patients with tumours due to multiple endocrine neoplasia type 2 and neurofibromatosis type 1 (NF1) showed similar catecholamine metabolite and secretory profiles to patients with adrenaline-producing tumours and no evident hereditary background. Tumours from these three patient groups contained higher contents of catecholamines, but secreted the hormones at lower rates than tumours that did not contain appreciable adrenaline, the latter including PPGLs due to von Hippel-Lindau (VHL) and succinate dehydrogenase (SDH) gene mutations. Large increases of plasma dopamine and its metabolites additionally characterised patients with PPGLs due to the latter mutations, whereas patients with NF1 were characterised by large increases in plasma dihydroxyphenylglycol and dihydroxyphenylacetic acid, the deaminated metabolites of noradrenaline and dopamine. This analysis establishes the utility of comprehensive catecholamine metabolite profiling for characterising the distinct and highly diverse catecholamine metabolomic and secretory phenotypes among different groups of patients with PPGLs. The data further suggest developmental origins of PPGLs from different populations of chromaffin cell progenitors.
Full-text · Article · Nov 2010 · Endocrine Related Cancer