Identification of potential molecular targets for the treatment of cluster 1 human pheochromocytoma and paraganglioma via comprehensive proteomic characterization.

BACKGROUND: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors. New drug targets and proteins that would assist sensitive PPGL imagining could improve therapy and quality of life of patients with PPGL, namely those with recurrent or metastatic disease. Using a combined proteomic strategy, we looked for such clinically relevant targets among integral membrane proteins (IMPs) upregulated on the surface of tumor cells and non-membrane druggable enzymes in PPGL. METHODS: We conducted a detailed proteomic analysis of 22 well-characterized human PPGL samples and normal chromaffin tissue from adrenal medulla. A standard quantitative proteomic analysis of tumor lysate, which provides information largely on non-membrane proteins, was accompanied by specific membrane proteome-aimed methods, namely glycopeptide enrichment using lectin-affinity, glycopeptide capture by hydrazide chemistry, and enrichment of membrane-embedded hydrophobic transmembrane segments. RESULTS: The study identified 67 cell surface integral membrane proteins strongly upregulated in PPGL compared to control chromaffin tissue. We prioritized the proteins based on their already documented direct role in cancer cell growth or progression. Increased expression of the seven most promising drug targets (CD146, CD171, ANO1, CD39, ATP8A1, ACE and SLC7A1) were confirmed using specific antibodies. Our experimental strategy also provided expression data for soluble proteins. Among the druggable non-membrane enzymes upregulated in PPGL, we identified three potential drug targets (SHMT2, ARG2 and autotaxin) and verified their upregulated expression. CONCLUSIONS: Application of a combined proteomic strategy recently presented as "Pitchfork" enabled quantitative analysis of both, membrane and non-membrane proteome, and resulted in identification of 10 potential drug targets in human PPGL. Seven membrane proteins localized on the cell surface and three non-membrane druggable enzymes proteins were identified and verified as significantly upregulated in PPGL. All the proteins have been previously shown to be upregulated in several human cancers, and play direct role in cancer progression. Marked upregulation of these proteins along with their localization and established direct roles in tumor progression make these molecules promising candidates as drug targets or proteins for sensitive PPGL imaging.

Autotaxin and Lysophosphatidic Acid Signalling: the Pleiotropic Regulatory Network in Cancer.

Autotaxin, also known as ecto-nucleotide pyrophosphatase/phosphodiesterase family member 2, is a secreted glycoprotein that plays multiple roles in human physiology and cancer pathology. This protein, by converting lysophosphatidylcholine into lysophosphatidic acid, initiates a complex signalling cascade with significant biological implications. The article outlines the autotaxin gene and protein structure, expression regulation and physiological functions, but focuses mainly on the role of autotaxin in cancer development and progression. Autotaxin and lysophosphatidic acid signalling influence several aspects of cancer, including cell proliferation, migration, metastasis, therapy resistance, and interactions with the immune system. The potential of autotaxin as a diagnostic biomarker and promising drug target is also examined.

Quantitative proteomic analysis of cerebrospinal fluid of women newly diagnosed with multiple sclerosis.

PURPOSE: The lack of reliable diagnostic and/or prognostic biomarkers for multiple sclerosis (MS) is the major obstacle to timely and accurate patient diagnosis in MS patients. To identify new proteins associated with MS we performed a detailed proteomic analysis of cerebrospinal fluid (CSF) of patients newly diagnosed with relapsing-remitting MS (RRMS) and healthy controls. MATERIAL: Reflecting significantly higher prevalence of MS in women we included only women patients and controls in the study. To eliminate a potential effect of therapy on the CSF composition, only the therapy-naïve patients were included. METHODS: Pooled CSF samples were processed in a technical duplicate, and labeled with stable-isotope coded TMT tags. To maximize the proteome coverage, peptide fractionation using 2D-LC preceded mass analysis using Orbitrap Fusion Tribrid Mass Spectrometer. Differential concentration of selected identified proteins between patients and controls was verified using specific antibodies. RESULTS: Of the identified 900 CSF proteins, we found 69 proteins to be differentially abundant between patients and controls. In addition to several proteins identified as differentially abundant in MS patients previously, we observed several linked to MS for the first time, namely eosinophil-derived neurotoxin and Nogo receptor. CONCLUSIONS: Our data confirm differential abundance of several previously proposed protein markers, and provide indirect support for involvement of copper-iron disbalance in MS. Most importantly, we identified two new differentially abundant CSF proteins that seem to be directly connected with myelin loss and axonal damage via TLR2 signaling and Nogo-receptor pathway in women newly diagnosed with RRMS.

Deep Membrane Proteome Profiling Reveals Overexpression of Prostate-Specific Membrane Antigen (PSMA) in High-Risk Human Paraganglioma and Pheochromocytoma, Suggesting New Theranostic Opportunity.

Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors arising from chromaffin cells of adrenal medulla or sympathetic or parasympathetic paraganglia, respectively. To identify new therapeutic targets, we performed a detailed membrane-focused proteomic analysis of five human paraganglioma (PGL) samples. Using the Pitchfork strategy, which combines specific enrichments of glycopeptides, hydrophobic transmembrane segments, and non-glycosylated extra-membrane peptides, we identified over 1800 integral membrane proteins (IMPs). We found 45 "tumor enriched" proteins, i.e., proteins identified in all five PGLs but not found in control chromaffin tissue. Among them, 18 IMPs were predicted to be localized on the cell surface, a preferred drug targeting site, including prostate-specific membrane antigen (PSMA), a well-established target for nuclear imaging and therapy of advanced prostate cancer. Using specific antibodies, we verified PSMA expression in 22 well-characterized human PPGL samples. Compared to control chromaffin tissue, PSMA was markedly overexpressed in high-risk PPGLs belonging to the established Cluster 1, which is characterized by worse clinical outcomes, pseudohypoxia, multiplicity, recurrence, and metastasis, specifically including SDHB, VHL, and EPAS1 mutations. Using immunohistochemistry, we localized PSMA expression to tumor vasculature. Our study provides the first direct evidence of PSMA overexpression in PPGLs which could translate to therapeutic and diagnostic applications of anti-PSMA radio-conjugates in high-risk PPGLs.

Affinity depletion versus relative protein enrichment: a side-by-side comparison of two major strategies for increasing human cerebrospinal fluid proteome coverage.

Cerebrospinal fluid (CSF) is in direct contact with the central nervous system. This makes human CSF an attractive source of potential biomarkers for neurologic diseases. Similarly to blood plasma, proteomic analysis of CSF is complicated by a high dynamic range of individual protein concentrations and by the presence of several highly abundant proteins. To deal with the abundant human CSF proteins, methods developed for blood plasma/serum are routinely used. Multiple affinity removal systems and protein enrichment of less abundant proteins using a combinatorial peptide ligand library are among the most frequent approaches. However, their relative impact on CSF proteome coverage has never been evaluated side-by-side in a single study. Therefore, we explored the effect of CSF depletion using MARS 14 cartridge and ProteoMiner ligand library on the number of CSF proteins identified in subsequent LC-MS/MS analysis. LC-MS/MS analysis of crude (non-treated) CSF provided roughly 500 identified proteins. Depletion of CSF by MARS 14 cartridge increased the number of identifications to nearly 800, while treatment of CSF using ProteoMiner enabled identification of 600 proteins. To explore the potential losses of CSF proteins during the depletion process, we also analyzed the "waste" fractions generated by both methods, i.e., proteins retained by the MARS 14 cartridge, and the molecules present in the flow-through fraction from ProteoMiner. More than 250 proteins were bound to MARS 14 cartridge, 100 of those were not identified in the corresponding depleted CSF. Similarly, analysis of the waste fraction in ProteoMiner workflow provided almost 70 unique proteins not found in the CSF depleted by the ligand library. Both depletion strategies significantly increased the number of identified CSF proteins compared to crude CSF. However, MARS 14 depletion provided a markedly higher number of identified proteins (773) compared to ProteoMiner (611). Further, we showed that CSF proteins are lost due to co-depletion (MARS 14) or exclusion (ProteoMiner) during the depletion process. This suggests that the routinely discarded "waste" fractions contain proteins of potential interest and should be included in CSF biomarker studies.

Kidney Response to Heart Failure: Proteomic Analysis of Cardiorenal Syndrome.

BACKGROUND/AIMS: Chronic heart failure (HF) disrupts normal kidney function and leads to cardiorenal syndrome that further promotes HF progression. To identify potential participants in HF-related injury, we analyzed kidney proteome in an established HF model. METHODS: HF was induced by chronic volume overload in male HanSD rats using aorto-caval fistula. After 21 weeks, cardiac and renal functions (in-situ kidney study) and renal proteomics were studied in sham-operated (controls) and HF rats, using iTRAQ labeling and LC-MS with Orbitrap Fusion, leading to identification and quantification of almost 4000 proteins. RESULTS: Compared to controls, HF rats had cardiac hypertrophy, systemic and pulmonary congestion. Kidneys of HF rats had reduced renal blood flow, sodium excretion and urine production. While glomerular filtration rate, serum cystatin C and creatinine were still normal compared to controls, HF kidneys showed albuminuria and markedly increased tissue angiotensin-II levels (5-fold). HF kidneys (versus controls) displayed differential expression (˃1.5-fold) of 67 proteins. The most upregulated were angiotensin-converting enzyme (ACE, ˃20-fold), advanced glycosylation product-specific receptor (RAGE, 14-fold), periostin (6.8-fold), caveolin-1 (4.5-fold) and other proteins implicated in endothelial function (vWF, cavins 1-3, T-kininogen 2), proinflammatory ECM activation (MFAP4, collagen-VI, galectin-3, FHL-1, calponin) and proteins involved in glomerular filtration membrane integrity (CLIC5, ZO-1). Carboxylesterase-1D (CES1D), an enzyme that converts ACE inhibitors or sacubitril into active drugs, was also upregulated in HF kidneys. CONCLUSION: Chronic HF leads to latent kidney injury, associated with deep changes in kidney protein composition. These alterations may act in concert with intrarenal renin-angiotensin system activation and may serve as markers and/or targets to tackle cardiorenal syndrome.

Downregulation of deoxycytidine kinase in cytarabine-resistant mantle cell lymphoma cells confers cross-resistance to nucleoside analogs gemcitabine, fludarabine and cladribine, but not to other classes of anti-lymphoma agents.

BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma associated with poor prognosis. Implementation of high-dose cytarabine (araC) into induction therapy became standard-of-care for all newly diagnosed younger MCL patients. However, many patients relapse even after araC-based regimen. Molecular mechanisms responsible for araC resistance in MCL are unknown and optimal treatment strategy for relapsed/refractory MCL patients remains elusive. METHODS: Five araC-resistant (R) clones were derived by long-term culture of five MCL cell lines (CTRL) with increasing doses of araC up to 50 microM. Illumina BeadChip and 2-DE proteomic analysis were used to identify gene and protein expression changes associated with araC resistance in MCL. In vitro cytotoxicity assays and experimental therapy of MCL xenografts in immunodeficient mice were used to analyze their relative responsiveness to a set of clinically used anti-MCL drugs. Primary MCL samples were obtained from patients at diagnosis and after failure of araC-based therapies. RESULTS: Marked downregulation of deoxycytidine-kinase (DCK) mRNA and protein expression was identified as the single most important molecular event associated with araC-resistance in all tested MCL cell lines and in 50% primary MCL samples. All R clones were highly (20-1000x) cross-resistant to all tested nucleoside analogs including gemcitabine, fludarabine and cladribine. In vitro sensitivity of R clones to other classes of clinically used anti-MCL agents including genotoxic drugs (cisplatin, doxorubicin, bendamustine) and targeted agents (bortezomib, temsirolimus, rituximab) remained unaffected, or was even increased (ibrutinib). Experimental therapy of immunodeficient mice confirmed the anticipated loss of anti-tumor activity (as determined by overall survival) of the nucleoside analogs gemcitabine and fludarabine in mice transplanted with R clone compared to mice transplanted with CTRL cells, while the anti-tumor activity of cisplatin, temsirolimus, bortezomib, bendamustine, cyclophosphamide and rituximab remained comparable between the two cohorts. CONCLUSIONS: Acquired resistance of MCL cells to araC is associated with downregulation of DCK, enzyme of the nucleotide salvage pathway responsible for the first phosphorylation (=activation) of most nucleoside analogs used in anti-cancer therapy. The data suggest that nucleoside analogs should not be used in the therapy of MCL patients, who relapse after failure of araC-based therapies.

Metabolomics and proteomics in pheochromocytoma and paraganglioma: Translating biochemistry and biology to bedside.

The complexity of omes - the key cellular ensembles (genome and epigenome, transcriptome, proteome, and metabolome) - is becoming increasingly understood in terms of big-data analysis, the omics. Amongst these, proteomics provides a global description of quantitative and qualitative alterations of protein expression (or protein abundance in body fluids) in response to physiologic or pathologic processes while metabolomics offers a functional portrait of the physiological state by quantifying metabolite abundances in biological samples. Here, we summarize how different techniques of proteomic and metabolic analysis can be used to define key biochemical characteristics of pheochromocytomas/paragangliomas (PPGL). The significance of omics in understanding features of PPGL biology that might translate to improved diagnosis and treatment will be highlighted.

Deciphering pathophysiological mechanisms underlying cystathionine beta-synthase-deficient homocystinuria using targeted metabolomics, liver proteomics, sphingolipidomics and analysis of mitochondrial function.

BACKGROUND: Cystathionine ß-synthase (CBS)-deficient homocystinuria (HCU) is an inherited disorder of sulfur amino acid metabolism with varying severity and organ complications, and a limited knowledge about underlying pathophysiological processes. Here we aimed at getting an in-depth insight into disease mechanisms using a transgenic mouse model of HCU (I278T). METHODS: We assessed metabolic, proteomic and sphingolipidomic changes, and mitochondrial function in tissues and body fluids of I278T mice and WT controls. Furthermore, we evaluated the efficacy of methionine-restricted diet (MRD) in I278T mice. RESULTS: In WT mice, we observed a distinct tissue/body fluid compartmentalization of metabolites with up to six-orders of magnitude differences in concentrations among various organs. The I278T mice exhibited the anticipated metabolic imbalance with signs of an increased production of hydrogen sulfide and disturbed persulfidation of free aminothiols. HCU resulted in a significant dysregulation of liver proteome affecting biological oxidations, conjugation of compounds, and metabolism of amino acids, vitamins, cofactors and lipids. Liver sphingolipidomics indicated upregulation of the pro-proliferative sphingosine-1-phosphate signaling pathway. Liver mitochondrial function of HCU mice did not seem to be impaired compared to controls. MRD in I278T mice improved metabolic balance in all tissues and substantially reduced dysregulation of liver proteome. CONCLUSION: The study highlights distinct tissue compartmentalization of sulfur-related metabolites in normal mice, extensive metabolome, proteome and sphingolipidome disruptions in I278T mice, and the efficacy of MRD to alleviate some of the HCU-related biochemical abnormalities.

FGF-23 is a biomarker of RV dysfunction and congestion in patients with HFrEF.

There is no biomarker reflecting right ventricular dysfunction in HFrEF patients used in clinical practice. We have aimed to look for a circulating marker of RV dysfunction employing a quantitative proteomic strategy. The Olink Proteomics Multiplex panels (Cardiovascular Disease II, III, Cardiometabolic, and Inflammation Target Panels) identified FGF-23 to be the most differentially abundant (more than 2.5-fold) in blood plasma of HF patients with severe RV dysfunction (n = 30) compared to those with preserved RV function (n = 31). A subsequent ELISA-based confirmatory analysis of circulating FGF-23 in a large cohort of patients (n = 344, 72.7% NYHA III/IV, LVEF 22.5%, 54.1% with moderate/severe RV dysfunction), followed by multivariable regression analysis, revealed that the plasma FGF-23 level was most significantly associated with RV dysfunction grade (p = 0.0004) and congestion in the systemic circulation (p = 0.03), but not with LV-ejection fraction (p = 0.69) or estimated glomerular filtration rate (eGFR, p = 0.08). FGF-23 was associated with the degree of RV dysfunction in both sub-cohorts (i.e. in patients with and without congestion, p < 0.0001). The association between FGF-23 and RV-dysfunction remained significant after the adjustment for BNP (p = 0.01). In contrast, when adjusted for BNP, FGF-23 was no longer associated with LV dysfunction (p = 0.59). The Cox proportional hazard model revealed that circulating FGF-23 was significantly associated with adverse outcomes even after adjusting for BNP, LVEF, RV dysfunction grade and eGFR. Circulating FGF-23 is thus a biomarker of right ventricular dysfunction in HFrEF patients regardless of congestion status.

Tyrosine 87 is vital for the activity of human protein arginine methyltransferase 3 (PRMT3).

Protein arginine methyltransferase 3 (PRMT3) is a cytosolic enzyme that catalyzes the formation of mono- and asymmetric dimethyl arginines, with ribosomal protein (RP) S2 as its main in vivo substrate. The interplay of PRMT3-RPS2 homologs in yeast is important for regulating the ribosomal subunit ratio and assembly. Prmt3-null mice display slower embryonic growth and development, although this phenotype is milder than in mouse RP gene knockouts. Defects in ribosome maturation are the hallmark of Diamond-Blackfan anemia (DBA). Sequencing of the PRMT3 gene in patients from the Czech DBA registry revealed a heterozygous mutation encoding the Tyr87Cys substitution. Although later analysis excluded this mutation as the cause of disease, we anticipated that this substitution might be important for PRMT3 function and decided to study it in detail. Tyr87 resides in a highly conserved substrate binding domain and has been predicted to be phosphorylated. To address the impact of putative Tyr87 phosphorylation on PRMT3 properties, we constructed two additional PRMT3 variants, Tyr87Phe and Tyr87Glu PRMT3, mimicking non-phosphorylated and phosphorylated Tyr87, respectively. The Tyr87Cys and Tyr87Glu-PRMT3 variants had markedly decreased affinity to RPS2 and, consequently, reduced enzymatic activity compared to the wild-type enzyme. The activity of the Tyr87Phe-PRMT3 mutant remained unaffected. No evidence of Tyr87 phosphorylation was found using mass spectrometric analysis of purified PRMT3, although phosphorylation of serines 25 and 27 was observed. In conclusion, Tyr87 is important for the interaction between PRMT3 and RPS2 and for its full enzymatic activity.

Nutritional hepatic iron overload is not prevented by parenteral hepcidin substitution therapy in mice.

The peptide hormone hepcidin functions as a negative regulator of intestinal Fe absorption and Fe recycling. Since its discovery as a systemic negative regulator of Fe metabolism, hepcidin has attracted enormous interest as a potential drug for the treatment and/or prevention of several forms of Fe overload. We therefore tested whether multiple doses of intraperitoneally administered synthetic renatured hepcidin can prevent hepatic Fe loading in mice concurrently fed an Fe-rich diet, and whether the same treatment affects hepatic Fe stores in mice fed a normal diet. Cohorts of male mice were fed either a normal defined diet (180 parts per million Fe) or an Fe-rich diet (the same diet supplemented with 2 % carbonyl iron for 2 weeks). Concurrently, half of the animals in each diet group received 100 µg of renatured hepcidin intraperitoneally every 12 h, for the same 2-week period. The second half of the animals received PBS only. The renatured synthetic hepcidin demonstrated biological activity by significantly decreasing transferrin saturation, which lasted for up to 24 h after a single hepcidin dose. However, the 14 d intraperitoneal hepcidin therapy did not prevent hepatic Fe overload in mice fed the Fe-rich diet, nor did it affect hepatic Fe stores in mice fed the normal diet. Both hepcidin agonists and antagonists are expected to have broad therapeutic potential. The absence of an effect of biologically active hepcidin on hepatic Fe loading shows the need for thorough future studies on the hepcidin regulation of Fe absorption and tissue distribution.

Flow Cytometry Analysis of Blood Large Extracellular Vesicles in Patients with Multiple Sclerosis Experiencing Relapse of the Disease.

The number of people living with multiple sclerosis (MS) in developed countries is increasing. The management of patients is hindered by the absence of reliable laboratory tests accurately reflecting the disease activity. Extracellular vesicles (EVs) of different cell origin were reportedly elevated in MS patients. We assessed the diagnostic potential, with flow cytometry analysis, of fresh large EVs (lEVs), which scattered more light than the 590 nm silica beads and were isolated from the blood plasma of relapsing remitting MS patients. Venous blood was collected from 15 patients and 16 healthy controls (HC). The lEVs were isolated from fresh platelet-free plasma by centrifugation, labelled with antibodies and the presence of platelet (CD41+, CD36+), endothelial (CD105+), erythrocyte (CD235a+), leukocyte (CD45+, CD19+, CD3+) and phosphatidylserine (Annexin V+) positive lEVs was analyzed using standard flow cytometry. Cryo-electron microscopy was used to verify the presence of EVs in the analyzed plasma fractions. MS patients experiencing acute relapse had slightly reduced relative levels (% of positive lEVs) of CD105+, CD45+, CD3+, CD45+CD3+ or CD19+ labelled lEVs in comparison to healthy controls. An analysis of other markers or a comparison of absolute lEV counts (count of lEVs/µL) did not yield any significant differences. Our data do not support the hypothesis that the exacerbation of the disease in RRMS patients leads to an increased numbers of circulating plasma lEVs which can be monitored by standard flow cytometry.

Hepcidin, the hormone of iron metabolism, is bound specifically to alpha-2-macroglobulin in blood.

Hepcidin is a major regulator of iron metabolism. Hepcidin-based therapeutics/diagnostics could play roles in hematology in the future, and thus, hepcidin transport is crucial to understand. In this study, we identify alpha2-macroglobulin (alpha2-M) as the specific hepcidin-binding molecule in blood. Interaction of 125I-hepcidin with alpha2-M was identified using fractionation of plasma proteins followed by native gradient polyacrylamide gel electrophoresis and mass spectrometry. Hepcidin binding to nonactivated alpha2-M displays high affinity (Kd 177 +/- 27 nM), whereas hepcidin binding to albumin was nonspecific and displayed nonsaturable kinetics. Surprisingly, the interaction of hepcidin with activated alpha2-M exhibited a classical sigmoidal binding curve demonstrating cooperative binding of 4 high-affinity (Kd 0.3 microM) hepcidin-binding sites. This property probably enables efficient sequestration of hepcidin and its subsequent release or inactivation that may be important for its effector functions. Because alpha2-M rapidly targets ligands to cells via receptor-mediated endocytosis, the binding of hepcidin to alpha2-M may influence its functions. In fact, the alpha2-M-hepcidin complex decreased ferroportin expression in J774 cells more effectively than hepcidin alone. The demonstration that alpha2-M is the hepcidin transporter could lead to better understanding of hepcidin physiology, methods for its sensitive measurement and the development of novel drugs for the treatment of iron-related diseases.

Effect of metformin therapy on cardiac function and survival in a volume-overload model of heart failure in rats.

Advanced HF (heart failure) is associated with altered substrate metabolism. Whether modification of substrate use improves the course of HF remains unknown. The antihyperglycaemic drug MET (metformin) affects substrate metabolism, and its use might be associated with improved outcome in diabetic HF. The aim of the present study was to examine whether MET would improve cardiac function and survival also in non-diabetic HF. Volume-overload HF was induced in male Wistar rats by creating ACF (aortocaval fistula). Animals were randomized to placebo/MET (300 mg·kg(-1) of body weight·day(-1), 0.5% in food) groups and underwent assessment of metabolism, cardiovascular and mitochondrial functions (n=6-12/group) in advanced HF stage (week 21). A separate cohort served for survival analysis (n=10-90/group). The ACF group had marked cardiac hypertrophy, increased LVEDP (left ventricular end-diastolic pressure) and lung weight confirming decompensated HF, increased circulating NEFAs (non-esterified 'free' fatty acids), intra-abdominal fat depletion, lower glycogen synthesis in the skeletal muscle (diaphragm), lower myocardial triacylglycerol (triglyceride) content and attenuated myocardial (14)C-glucose and (14)C-palmitate oxidation, but preserved mitochondrial respiratory function, glucose tolerance and insulin sensitivity. MET therapy normalized serum NEFAs, decreased myocardial glucose oxidation, increased myocardial palmitate oxidation, but it had no effect on myocardial gene expression, AMPK (AMP-activated protein kinase) signalling, ATP level, mitochondrial respiration, cardiac morphology, function and long-term survival, despite reaching therapeutic serum levels (2.2±0.7 µg/ml). In conclusion, MET-induced enhancement of myocardial fatty acid oxidation had a neutral effect on cardiac function and survival. Recently reported cardioprotective effects of MET may not be universal to all forms of HF and may require AMPK activation or ATP depletion. No increase in mortality on MET supports its safe use in diabetic HF.

Proteomic and transcriptomic analysis of heart failure due to volume overload in a rat aorto-caval fistula model provides support for new potential therapeutic targets - monoamine oxidase A and transglutaminase 2.

BACKGROUND: Chronic hemodynamic overloading leads to heart failure (HF) due to incompletely understood mechanisms. To gain deeper insight into the molecular pathophysiology of volume overload-induced HF and to identify potential markers and targets for novel therapies, we performed proteomic and mRNA expression analysis comparing myocardium from Wistar rats with HF induced by a chronic aorto-caval fistula (ACF) and sham-operated rats harvested at the advanced, decompensated stage of HF. METHODS: We analyzed control and failing myocardium employing iTRAQ labeling, two-dimensional peptide separation combining peptide IEF and nano-HPLC with MALDI-MS/MS. For the transcriptomic analysis we employed Illumina RatRef-12v1 Expression BeadChip. RESULTS: In the proteomic analysis we identified 2030 myocardial proteins, of which 66 proteins were differentially expressed. The mRNA expression analysis identified 851 differentially expressed mRNAs. CONCLUSIONS: The differentially expressed proteins confirm a switch in the substrate preference from fatty acids to other sources in the failing heart. Failing hearts showed downregulation of the major calcium transporters SERCA2 and ryanodine receptor 2 and altered expression of creatine kinases. Decreased expression of two NADPH producing proteins suggests a decreased redox reserve. Overexpression of annexins supports their possible potential as HF biomarkers. Most importantly, among the most up-regulated proteins in ACF hearts were monoamine oxidase A and transglutaminase 2 that are both potential attractive targets of low molecular weight inhibitors in future HF therapy.

Metabolic characterization of volume overload heart failure due to aorto-caval fistula in rats.

Metabolic interactions between adipose tissue and the heart may play an active role in progression of heart failure (HF). The aim of the study was to examine changes in myocardial and adipose tissue metabolism and gene expression in a rat HF model induced by chronic volume overload. HF was induced by volume overload from aorto-caval fistula (ACF) in 3-month-old male Wistar rats and animals were studied in the phase of decompensated HF (22nd week). HF rats showed marked eccentric cardiac hypertrophy, pulmonary congestion, increased LV end-diastolic pressure, and intraabdominal fat depletion. HF rats had preserved glucose tolerance, but increased circulating free fatty acids (FFA) and attenuated insulin response during oral glucose challenge. Isolated organ studies showed preserved responsiveness of adipose tissue lipolysis and lipogenesis to epinephrine and insulin in ACF. The heart of HF animals had markedly reduced triglyceride content (almost to half of controls), attenuated anti-oxidative reserve (GSH/GSSG), upregulated HF markers (ANP, periostin, thrombospondin-4), specific signaling pathways (Wnt, TGF-ß), and downregulated enzymes of mitochondrial fatty acid oxidation, citric acid cycle, and respiratory chain. Adipose tissue transcription profiling showed upregulated receptor for gastric inhibitory polypeptide. In conclusion, ACF-induced HF model displays several deregulations of systemic metabolism. Despite elevation of systemic FFAs, myocardial triglycerides are low and insulin levels are attenuated, arguing against a role of lipotoxicity or insulin resistance in this model. Attenuated postprandial insulin response and relative lack of its antilipolytic effects may facilitate intraabdominal fat depletion observed in ACF-HF animals.

Right versus left ventricular remodeling in heart failure due to chronic volume overload.

Mechanisms of right ventricular (RV) dysfunction in heart failure (HF) are poorly understood. RV response to volume overload (VO), a common contributing factor to HF, is rarely studied. The goal was to identify interventricular differences in response to chronic VO. Rats underwent aorto-caval fistula (ACF)/sham operation to induce VO. After 24 weeks, RV and left ventricular (LV) functions, gene expression and proteomics were studied. ACF led to biventricular dilatation, systolic dysfunction and hypertrophy affecting relatively more RV. Increased RV afterload contributed to larger RV stroke work increment compared to LV. Both ACF ventricles displayed upregulation of genes of myocardial stress and metabolism. Most proteins reacted to VO in a similar direction in both ventricles, yet the expression changes were more pronounced in RV (pslope: < 0.001). The most upregulated were extracellular matrix (POSTN, NRAP, TGM2, CKAP4), cell adhesion (NCAM, NRAP, XIRP2) and cytoskeletal proteins (FHL1, CSRP3) and enzymes of carbohydrate (PKM) or norepinephrine (MAOA) metabolism. Downregulated were MYH6 and FAO enzymes. Therefore, when exposed to identical VO, both ventricles display similar upregulation of stress and metabolic markers. Relatively larger response of ACF RV compared to the LV may be caused by concomitant pulmonary hypertension. No evidence supports RV chamber-specific regulation of protein expression in response to VO.

Exosomes released by imatinib­resistant K562 cells contain specific membrane markers, IFITM3, CD146 and CD36 and increase the survival of imatinib­sensitive cells in the presence of imatinib.

Chronic myeloid leukemia (CML) is a malignant hematopoietic disorder distinguished by the presence of a BCR­ABL1 fused oncogene with constitutive kinase activity. Targeted CML therapy by specific tyrosine kinase inhibitors (TKIs) leads to a marked improvement in the survival of the patients and their quality of life. However, the development of resistance to TKIs remains a critical issue for a subset of patients. The most common cause of resistance are numerous point mutations in the BCR­ABL1 gene, followed by less common mutations and multiple mutation-independent mechanisms. Recently, exosomes, which are extracellular vesicles excreted from normal and tumor cells, have been associated with drug resistance and cancer progression. The aim of the present study was to characterize the exosomes released by imatinib­resistant K562 (K562IR) cells. The K562IR­derived exosomes were internalized by imatinib­sensitive K562 cells, which thereby increased their survival in the presence of 2 µM imatinib. The exosomal cargo was subsequently analyzed to identify resistance­associated markers using a deep label­free quantification proteomic analysis. There were >3,000 exosomal proteins identified of which, 35 were found to be differentially expressed. From this, a total of 3, namely the membrane proteins, interferon­induced transmembrane protein 3, CD146 and CD36, were markedly upregulated in the exosomes derived from the K562IR cells, and exhibited surface localization. The upregulation of these proteins was verified in the K562IR exosomes, and also in the K562IR cells. Using flow cytometric analysis, it was possible to further demonstrate the potential of CD146 as a cell surface marker associated with imatinib resistance in K562 cells. Taken together, these results suggested that exosomes and their respective candidate surface proteins could be potential diagnostic markers of TKI drug resistance in CML therapy.

Human MRCKalpha is regulated by cellular iron levels and interferes with transferrin iron uptake.

Myotonic dystrophy kinase-related Cdc42-binding kinase alpha (MRCKalpha, formally known as CDC42BPA) is a serine/threonine kinase that can regulate actin/myosin assembly and activity. Recently, it has been shown that it possesses a functional iron responsive element (IRE) in the 3'-untranslated region (UTR) of its mRNA, suggesting that it may be involved in iron metabolism. Here we report that MRCKalpha protein expression is also regulated by iron levels; MRCKalpha colocalizes with transferrin (Tf)-loaded transferrin receptors (TfR), and attenuation of MRCKalpha expression by a short hairpin RNA silencing construct leads to a significant decrease in Tf-mediated iron uptake. Our results thus indicate that MRCKalpha takes part in Tf-iron uptake, probably via regulation of Tf-TfR endocytosis/endosome trafficking that is dependent on the cellular cytoskeleton. Regulation of the MRCKalpha activity by intracellular iron levels could thus represent another molecular feedback mechanism cells could use to finely tune iron uptake to actual needs.