Identification of myeloid-derived growth factor as a mechanically-induced, growth-promoting angiocrine signal for human hepatocytes.

Recently, we have shown that after partial hepatectomy (PHx), an increased hepatic blood flow initiates liver growth in mice by vasodilation and mechanically-triggered release of angiocrine signals. Here, we use mass spectrometry to identify a mechanically-induced angiocrine signal in human hepatic endothelial cells, that is, myeloid-derived growth factor (MYDGF). We show that it induces proliferation and promotes survival of primary human hepatocytes derived from different donors in two-dimensional cell culture, via activation of mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3). MYDGF also enhances proliferation of human hepatocytes in three-dimensional organoids. In vivo, genetic deletion of MYDGF decreases hepatocyte proliferation in the regenerating mouse liver after PHx; conversely, adeno-associated viral delivery of MYDGF increases hepatocyte proliferation and MAPK signaling after PHx. We conclude that MYDGF represents a mechanically-induced angiocrine signal and that it triggers growth of, and provides protection to, primary mouse and human hepatocytes.

The impact of mild hypoxia exposure on myokine secretion in human obesity.

BACKGROUND/OBJECTIVE: Compelling evidence indicates that myokines act in an autocrine, paracrine and endocrine manner to alter metabolic homeostasis. The mechanisms underlying exercise-induced changes in myokine secretion remain to be elucidated. Since exercise acutely decreases oxygen partial pressure (pO2) in skeletal muscle (SM), the present study was designed to test the hypothesis that (1) hypoxia exposure impacts myokine secretion in primary human myotubes and (2) exposure to mild hypoxia in vivo alters fasting and postprandial plasma myokine concentrations in humans. METHODS: Differentiated primary human myotubes were exposed to different physiological pO2 levels for 24 h, and cell culture medium was harvested to determine myokine secretion. Furthermore, we performed a randomized single-blind crossover trial to investigate the impact of mild intermittent hypoxia exposure (MIH: 7-day exposure to 15% O2, 3x2h/day vs. normoxia: 21% O2) on in vivo SM pO2 and plasma myokine concentrations in 12 individuals with overweight and obesity (body-mass index ≥ 28 kg/m2). RESULTS: Hypoxia exposure (1% O2) increased secreted protein acidic and rich in cysteine (SPARC, p = 0.043) and follistatin like 1 (FSTL1, p = 0.021), and reduced leukemia inhibitory factor (LIF) secretion (p = 0.009) compared to 3% O2 in primary human myotubes. In addition, 1% O2 exposure increased interleukin-6 (IL-6, p = 0.004) and SPARC secretion (p = 0.021), whilst reducing fatty acid binding protein 3 (FABP3) secretion (p = 0.021) compared to 21% O2. MIH exposure in vivo markedly decreased SM pO2 (≈40%, p = 0.002) but did not alter plasma myokine concentrations. CONCLUSIONS: Hypoxia exposure altered the secretion of several myokines in primary human myotubes, revealing hypoxia as a novel modulator of myokine secretion. However, both acute and 7-day MIH exposure did not induce alterations in plasma myokine concentrations in individuals with overweight and obesity. CLINICAL TRIALS IDENTIFIER: This study is registered at the Netherlands Trial Register (NL7120/NTR7325).

Protein Expression of AEBP1, MCM4, and FABP4 Differentiate Osteogenic, Adipogenic, and Mesenchymal Stromal Stem Cells.

Mesenchymal stem cells (MSCs) gain an increasing focus in the field of regenerative medicine due to their differentiation abilities into chondrocytes, adipocytes, and osteoblastic cells. However, it is apparent that the transformation processes are extremely complex and cause cellular heterogeneity. The study aimed to characterize differences between MSCs and cells after adipogenic (AD) or osteoblastic (OB) differentiation at the proteome level. Comparative proteomic profiling was performed using tandem mass spectrometry in data-independent acquisition mode. Proteins were quantified by deep neural networks in library-free mode and correlated to the Molecular Signature Database (MSigDB) hallmark gene set collections for functional annotation. We analyzed 4108 proteins across all samples, which revealed a distinct clustering between MSCs and cell differentiation states. Protein expression profiling identified activation of the Peroxisome proliferator-activated receptors (PPARs) signaling pathway after AD. In addition, two distinct protein marker panels could be defined for osteoblastic and adipocytic cell lineages. Hereby, overexpression of AEBP1 and MCM4 for OB as well as of FABP4 for AD was detected as the most promising molecular markers. Combination of deep neural network and machine-learning algorithms with data-independent mass spectrometry distinguish MSCs and cell lineages after adipogenic or osteoblastic differentiation. We identified specific proteins as the molecular basis for bone formation, which could be used for regenerative medicine in the future.

Oral Treatment with RD2RD2 Impedes Development of Motoric Phenotype and Delays Symptom Onset in SOD1G93A Transgenic Mice.

Neuroinflammation is a pathological hallmark of several neurodegenerative disorders and plays a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS). It has been implicated as driver of disease progression and is observed in ALS patients, as well as in the transgenic SOD1G93A mouse model. Here, we explore and validate the therapeutic potential of the d-enantiomeric peptide RD2RD2 upon oral administration in SOD1G93A mice. Transgenic mice were treated daily with RD2RD2 or placebo for 10 weeks and phenotype progression was followed with several behavioural tests. At the end of the study, plasma cytokine levels and glia cell markers in brain and spinal cord were analysed. Treatment resulted in a significantly increased performance in behavioural and motor coordination tests and a decelerated neurodegenerative phenotype in RD2RD2-treated SOD1G93A mice. Additionally, we observed retardation of the average disease onset. Treatment of SOD1G93A mice led to significant reduction in glial cell activation and a rescue of neurons. Analysis of plasma revealed normalisation of several cytokines in samples of RD2RD2-treated SOD1G93A mice towards the levels of non-transgenic mice. In conclusion, these findings qualify RD2RD2 to be considered for further development and testing towards a disease modifying ALS treatment.

Preparation of "Functional" Mitochondria: A Challenging Business.

As the powerhouse of the cell, mitochondria, plays a crucial role in many aspects of life, whereby mitochondrial dysfunctions are associated with pathogenesis of many diseases, like neurodegenerative diseases, obesity, cancer, and metabolic as well as cardiovascular disorders. Mitochondria analysis frequently starts with isolation and enrichment procedures, which have become increasingly important in biomedical research. Unfortunately, isolation procedures can easily cause changes in the structural integrity of mitochondria during in vitro handling having impact on their function. This carries the risk that conclusions about isolated mitochondria may be drawn on the basis of experimental artifacts. Here we critically review a commonly used isolation procedure for mitochondria utilizing differential (gradient) centrifugation and depict major challenges to achieve "functional" mitochondria as basis for comprehensive physiological studies.

AKT/AMPK-mediated phosphorylation of TBC1D4 disrupts the interaction with insulin-regulated aminopeptidase.

TBC1D4 is a 160 kDa multidomain Rab GTPase-activating protein (RabGAP) and a downstream target of the insulin- and contraction-activated kinases AKT and AMPK. Phosphorylation of TBC1D4 has been linked to translocation of GLUT4 from storage vesicles (GSVs) to the cell surface. However, its impact on enzymatic activity is not well understood, as previous studies mostly investigated the truncated GAP domain lacking the known phosphorylation sites. In the present study, we expressed and purified recombinant full-length TBC1D4 using a baculovirus system. Size-exclusion chromatography and coimmunoprecipitation experiments revealed that full-length TBC1D4 forms oligomers of ∼600 kDa. Compared with the truncated GAP domain, full-length TBC1D4 displayed similar substrate specificity, but had a markedly higher specific GAP activity toward Rab10. Using high-resolution mass spectrometry, we mapped 19 Ser/Thr phosphorylation sites in TBC1D4. We determined Michaelis-Menten kinetics using in vitro phosphorylation assays with purified kinases and stable isotope-labeled γ-[18O4]-ATP. These data revealed that Ser324 (KM ∼6 µM) and Thr649 (KM ∼25 µM) were preferential sites for phosphorylation by AKT, whereas Ser348, Ser577, Ser595 (KM ∼10 µM), Ser711 (KM ∼79 µM), and Ser764 were found to be preferred targets for AMPK. Phosphorylation of TBC1D4 by AKT or AMPK did not alter the intrinsic RabGAP activity, but did disrupt interaction with insulin-regulated aminopeptidase (IRAP), a resident protein of GSVs implicated in GLUT4 trafficking. These findings provide evidence that insulin and contraction may regulate TBC1D4 function primarily by disrupting the recruitment of the RabGAP to GLUT4 vesicles.

Protein Profiling of Serum Extracellular Vesicles Reveals Qualitative and Quantitative Differences After Differential Ultracentrifugation and ExoQuickTM Isolation.

Solid tumor biopsies are the current standard for precision medicine. However, the procedure is invasive and not always feasible. In contrast, liquid biopsies, such as serum enriched for extracellular vesicles (EVs) represent a non-invasive source of cancer biomarkers. In this study, we compared two EV isolation methods in the context of the protein biomarker detection in inflammatory bowel disease (IBD) and colorectal cancer (CRC). Using serum samples of a healthy cohort as well as CRC and IBD patients, EVs were isolated by ultracentrifugation and ExoQuickTM in parallel. EV associated protein profiles were compared by multiplex-fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) and subsequent identification by mass spectrometry. Validation of gelsolin (GSN) was performed using fluorescence-quantitative western blot. 2D-DIGE resolved 936 protein spots in all serum-enriched EVs isolated by ultracentrifugation or ExoQuickTM. Hereof, 93 spots were differently expressed between isolation approaches. Higher levels of GSN in EVs obtained with ExoQuickTM compared to ultracentrifugation were confirmed by western blot (p = 0.0006). Although patient groups were distinguishable after both EV isolation approaches, sample preparation strongly influences EVs' protein profile and thus impacts on inter-study reproducibility, biomarker identification and validation. The results stress the need for strict SOPs in EV research before clinical implementation can be reached.

Rhein, a novel Histone Deacetylase (HDAC) inhibitor with antifibrotic potency in human myocardial fibrosis.

Although fibrosis depicts a reparative mechanism, maladaptation of the heart due to excessive production of extracellular matrix accelerates cardiac dysfunction. The anthraquinone Rhein was examined for its anti-fibrotic potency to mitigate cardiac fibroblast-to-myofibroblast transition (FMT). Primary human ventricular cardiac fibroblasts were subjected to hypoxia and characterized with proteomics, transcriptomics and cell functional techniques. Knowledge based analyses of the omics data revealed a modulation of fibrosis-associated pathways and cell cycle due to Rhein administration during hypoxia, whereas p53 and p21 were identified as upstream regulators involved in the manifestation of cardiac fibroblast phenotypes. Mechanistically, Rhein acts inhibitory on HDAC classes I/II as enzymatic inhibitor. Rhein-mediated cellular effects were linked to the histone deacetylase (HDAC)-dependent protein stabilization of p53 under normoxic but not hypoxic conditions. Functionally, Rhein inhibited collagen contraction, indicating anti-fibrotic property in cardiac remodeling. This was accompanied by increased abundance of SMAD7, but not SMAD2/3, and consistently SMAD-specific E3 ubiquitin ligase SMURF2. In conclusion, this study identifies Rhein as a novel potent direct HDAC inhibitor that may contribute to the treatment of cardiac fibrosis as anti-fibrotic agent. As readily available drug with approved safety, Rhein constitutes a promising potential therapeutic approach in the supplemental and protective intervention of cardiac fibrosis.

DPP4 deletion in adipose tissue improves hepatic insulin sensitivity in diet-induced obesity.

Besides a therapeutic target for type 2 diabetes, dipeptidyl peptidase 4 (DPP4) is an adipokine potentially upregulated in human obesity. We aimed to explore the role of adipocyte-derived DPP4 in diet-induced obesity and insulin resistance with an adipose tissue-specific knockout (AT-DPP4-KO) mouse. Wild-type and AT-DPP4-KO mice were fed for 24 wk with a high fat diet (HFD) and characterized for body weight, glucose tolerance, insulin sensitivity by hyperinsulinemic-euglycemic clamp, and body composition and hepatic fat content. Image and molecular biology analysis of inflammation, as well as adipokine secretion, was performed in AT by immunohistochemistry, Western blot, real-time-PCR, and ELISA. Incretin levels were determined by Luminex kits. Under HFD, AT-DPP4-KO displayed markedly reduced circulating DPP4 concentrations, proving AT as a relevant source. Independently of glucose-stimulated incretin hormones, AT-DPP4-KO had improved glucose tolerance and hepatic insulin sensitivity. AT-DPP4-KO displayed smaller adipocytes and increased anti-inflammatory markers. IGF binding protein 3 (IGFBP3) levels were lower in AT and serum, whereas free IGF1 was increased. The absence of adipose DPP4 triggers beneficial AT remodeling with decreased production of IGFBP3 during HFD, likely contributing to the observed, improved hepatic insulin sensitivity.

Identification of the Secreted Proteins Originated from Primary Human Hepatocytes and HepG2 Cells.

The liver plays a pivotal role in whole-body carbohydrate, lipid, and protein metabolism. One of the key regulators of glucose and lipid metabolism are hepatokines, which are found among the liver secreted proteins, defined as liver secretome. To elucidate the composition of the human liver secretome and identify hepatokines in primary human hepatocytes (PHH), we conducted comprehensive protein profiling on conditioned medium (CM) of PHH. Secretome profiling using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) identified 691 potential hepatokines in PHH. Subsequently, pathway analysis assigned these proteins to acute phase response, coagulation, and complement system pathways. The secretome of PHH was compared to the secreted proteins of the liver hepatoma cell line HepG2. Although the secretome of PHH and HepG2 cells show a high overlap, the HepG2 secretome rather mirrors the fetal liver with some cancer characteristics. Collectively, our study represents one of the most comprehensive secretome profiling approaches for PHH, allowing new insights into the composition of the secretome derived from primary human material, and points out strength and weakness of using HepG2 cell secretome as a model for the analysis of the human liver secretome.

Decreased M1 macrophage polarization in dabigatran-treated Ldlr-deficient mice: Implications for atherosclerosis and adipose tissue inflammation.

BACKGROUND AND AIMS: The non-vitamin K oral anticoagulant dabigatran etexilate (dabigatran) is increasingly prescribed to patients with non-valvular atrial fibrillation and venous thromboembolism. Adipose tissue (AT) inflammation during obesity plays a crucial role in the development of insulin resistance, type II diabetes and atherogenesis. The aim of the present study was to investigate the effects of thrombin inhibition by dabigatran in a combined model of diet-induced obesity and atherosclerosis. METHODS: Female Low density lipoprotein receptor knockout (Lldr-/-) mice were fed a high-fat diet containing 5 mg/g dabigatran or matching control for 20 weeks. RESULTS: Dabigatran-treated animals showed increased adipocyte hypertrophy, but reduced numbers of pro-inflammatory M1-polarized macrophages in the adipose tissue. Abundance of pro-inflammatory M1 macrophages was also decreased in the aortic wall of dabigatran-fed mice. Multiple circulating cytokines were reduced, indicating an effect in systemically relevant secretory compartments such as the AT. CONCLUSIONS: Dabigatran treatment reduces pro-inflammatory M1 macrophages in atherosclerotic lesions, thereby contributing to plaque stabilizing and atheroprotective effects of the thrombin inhibitor. This finding is not restricted to the vascular wall but is also present in AT where dabigatran treatment reduced the release of pro-inflammatory cytokines and accumulation of M1 macrophages.

Cardiac Hyaluronan Synthesis Is Critically Involved in the Cardiac Macrophage Response and Promotes Healing After Ischemia Reperfusion Injury.

RATIONALE: Immediate changes in the ECM (extracellular matrix) microenvironment occur after myocardial ischemia and reperfusion (I/R) injury. OBJECTIVE: Aim of this study was to unravel the role of the early hyaluronan (HA)-rich ECM after I/R. METHODS AND RESULTS: Genetic deletion of Has2 and Has1 was used in a murine model of cardiac I/R. Chemical exchange saturation transfer imaging was adapted to image cardiac ECM post-I/R. Of note, the cardiac chemical exchange saturation transfer signal was severely suppressed by Has2 deletion and pharmacological inhibition of HA synthesis 24 hours after I/R. Has2 KO ( Has2 deficient) mice showed impaired hemodynamic function suggesting a protective role for endogenous HA synthesis. In contrast to Has2 deficiency, Has1-deficient mice developed no specific phenotype compared with control post-I/R. Importantly, in Has2 KO mice, cardiac macrophages were diminished after I/R as detected by 19F MRI (magnetic resonance imaging) of perfluorcarbon-labeled immune cells, Mac-2/Galectin-3 immunostaining, and FACS (fluorescence-activated cell sorting) analysis (CD45+CD11b+Ly6G-CD64+F4/80+cells). In contrast to macrophages, cardiac Ly6Chigh and Ly6Clow monocytes were unaffected post-I/R compared with control mice. Mechanistically, inhibition of HA synthesis led to increased macrophage apoptosis in vivo and in vitro. In addition, α-SMA (α-smooth muscle actin)-positive cells were reduced in the infarcted myocardium and in the border zone. In vitro, the myofibroblast response as measured by Acta2 mRNA expression was reduced by inhibition of HA synthesis and of CD44 signaling. Furthermore, Has2 KO fibroblasts were less able to contract collagen gels in vitro. The effects of HA/CD44 on fibroblasts and macrophages post-I/R might also affect intercellular cross talk because cardiac fibroblasts were activated by monocyte/macrophages and, in turn, protected macrophages from apoptosis. CONCLUSIONS: Increased HA synthesis contributes to postinfarct healing by supporting macrophage survival and by promoting the myofibroblast response. Additionally, imaging of cardiac HA by chemical exchange saturation transfer post-I/R might have translational value.

Aß Oligomer Elimination Restores Cognition in Transgenic Alzheimer's Mice with Full-blown Pathology.

Oligomers of the amyloid-ß (Aß) protein are suspected to be responsible for the development and progression of Alzheimer's disease. Thus, the development of compounds that are able to eliminate already formed toxic Aß oligomers is very desirable. Here, we describe the in vivo efficacy of the compound RD2, which was developed to directly and specifically eliminate toxic Aß oligomers. In a truly therapeutic, rather than a preventive study, oral treatment with RD2 was able to reverse cognitive deficits and significantly reduce Aß pathology in old-aged transgenic Alzheimer's Disease mice with full-blown pathology and behavioral deficits. For the first time, we demonstrate the in vivo target engagement of RD2 by showing a significant reduction of Aß oligomers in the brains of RD2-treated mice compared to placebo-treated mice. The correlation of Aß elimination in vivo and the reversal of cognitive deficits in old-aged transgenic mice support the hypothesis that Aß oligomers are relevant not only for disease development and progression, but also offer a promising target for the causal treatment of Alzheimer's disease.

AKT and AMP-activated protein kinase regulate TBC1D1 through phosphorylation and its interaction with the cytosolic tail of insulin-regulated aminopeptidase IRAP.

In skeletal muscle, the Rab GTPase-activating (GAP) protein TBC1D1 is phosphorylated by AKT and AMP-activated protein kinase (AMPK) in response to insulin and muscle contraction. Genetic ablation of Tbc1d1 or mutation of distinct phosphorylation sites impairs intracellular GLUT4 retention and GLUT4 traffic, presumably through alterations of the activation state of downstream Rab GTPases. Previous studies have focused on characterizing the C-terminal GAP domain of TBC1D1 that lacks the known phosphorylation sites, as well as putative regulatory domains. As a result, it has been unclear how phosphorylation of TBC1D1 would regulate its activity. In the present study, we have expressed, purified, and characterized recombinant full-length TBC1D1 in Sf9 insect cells via the baculovirus system. Full-length TBC1D1 showed RabGAP activity toward GLUT4-associated Rab8a, Rab10, and Rab14, indicating similar substrate specificity as the truncated GAP domain. However, the catalytic activity of the full-length TBC1D1 was markedly higher than that of the GAP domain. Although in vitro phosphorylation of TBC1D1 by AKT or AMPK increased 14-3-3 binding, it did not alter the intrinsic RabGAP activity. However, we found that TBC1D1 interacts through its N-terminal PTB domains with the cytoplasmic domain of the insulin-regulated aminopeptidase, a resident protein of GLUT4 storage vesicles, and this binding is disrupted by phosphorylation of TBC1D1 by AKT or AMPK. In summary, our findings suggest that other regions outside the GAP domain may contribute to the catalytic activity of TBC1D1. Moreover, our data indicate that recruitment of TBC1D1 to GLUT4-containing vesicles and not its GAP activity is regulated by insulin and contraction-mediated phosphorylation.

Protein levels of clusterin and glutathione synthetase in platelets allow for early detection of colorectal cancer.

Colorectal cancer (CRC) is one of the most frequent malignancies in the Western world. Early tumor detection and intervention are important determinants on CRC patient survival. During early tumor proliferation, dissemination and angiogenesis, platelets store and segregate proteins actively and selectively. Hence, the platelet proteome is a potential source of biomarkers denoting early malignancy. By comparing protein profiles of platelets between healthy volunteers (n = 12) and patients with early- (n = 7) and late-stage (n = 5) CRCs using multiplex fluorescence two-dimensional gel electrophoresis (2D-DIGE), we aimed at identifying differentially regulated proteins within platelets. By inter-group comparisons, 94 differentially expressed protein spots were detected (p < 0.05) between healthy controls and patients with early- and late-stage CRCs and revealed distinct separations between all three groups in principal component analyses. 54 proteins of interest were identified by mass spectrometry and resulted in high-ranked Ingenuity Pathway Analysis networks associated with Cellular function and maintenance, Cellular assembly and organization, Developmental disorder and Organismal injury and abnormalities (p < 0.0001 to p = 0.0495). Target proteins were validated by multiplex fluorescence-based Western blot analyses using an additional, independent cohort of platelet protein samples [healthy controls (n = 15), early-stage CRCs (n = 15), late-stage CRCs (n = 15)]. Two proteins-clusterin and glutathione synthetase (GSH-S)-featured high impact and were subsequently validated in this independent clinical cohort distinguishing healthy controls from patients with early- and late-stage CRCs. Thus, the potential of clusterin and GSH-S as platelet biomarkers for early detection of CRC could improve existing screening modalities in clinical application and should be confirmed in a prospective multicenter trial.

EB1 protein alteration characterizes sporadic but not ulcerative colitis associated colorectal cancer.

BACKGROUND: While carcinogenesis in Sporadic Colorectal Cancer (SCC) has been thoroughly studied, less is known about Ulcerative Colitis associated Colorectal Cancer (UCC). This study aimed to identify and validate differentially expressed proteins between clinical samples of SCC and UCC to elucidate new insights of UCC/SCC carcinogenesis and progression. RESULTS: Multiplex-fluorescence two-dimensional gel electrophoresis (2-D DIGE) and mass spectrometry identified 67 proteoforms representing 43 distinct proteins. After analysis by Ingenuity Pathway Analysis® (IPA), subsequent Western blot validation proofed the differential expression of Heat shock 27 kDA protein 1 (HSPB1) and Microtubule-associated protein R/EB family, member 1 (EB1) while the latter one showed also expression differences by immunohistochemistry. MATERIALS AND METHODS: Fresh frozen tissue of UCC (n = 10) matched with SCC (n = 10) was investigated. Proteins of cancerous intestinal mucosal cells were obtained by Laser Capture Microdissection (LCM) and compared by 2-D DIGE. Significant spots were identified by mass spectrometry. After IPA, three proteins [EB1, HSPB1, and Annexin 5 (ANXA5)] were chosen for further validation by Western blotting and tissue microarray-based immunohistochemistry. CONCLUSIONS: This study identified significant differences in protein expression of colorectal carcinoma cells from UCC patients compared to patients with SCC. Particularly, EB1 was validated in an independent clinical cohort.

Oxygen and differentiation status modulate the effect of X-ray irradiation on physiology and mitochondrial proteome of human neuroblastoma cells.

Cytotoxic effects, including oxidative stress, of low linear energy transfer (LET)-ionizing radiation are often underestimated and studies of their mechanisms using cell culture models are widely conducted with cells cultivated at atmospheric oxygen that does not match its physiological levels in body tissues. Also, cell differentiation status plays a role in the outcome of experiments. We compared effects of 2 Gy X-ray irradiation on the physiology and mitochondrial proteome of nondifferentiated and human neuroblastoma (SH-SY5Y) cells treated with retinoic acid cultivated at 21% and 5% O2. Irradiation did not affect the amount of subunits of OxPhos complexes and other non-OxPhos mitochondrial proteins, except for heat shock protein 70, which was increased depending on oxygen level and differentiation status. These two factors were proven to modulate mitochondrial membrane potential and the bioenergetic status of cells. We suggest, moreover, that oxygen plays a role in the differentiation of human SH-SY5Y cells.

Deletion of Hyaluronan Synthase 3 Inhibits Neointimal Hyperplasia in Mice.

OBJECTIVE: Hyaluronan (HA) is a polymeric glucosaminoglycan that forms a provisional extracellular matrix in diseased vessels. HA is synthesized by 3 different HA synthases (HAS1, HAS2, and HAS3). Aim of this study was to unravel the role of the HAS3 isoenzyme during experimental neointimal hyperplasia. APPROACH AND RESULTS: Neointimal hyperplasia was induced in Has3-deficient mice by ligation of the carotid artery. HA in the media of Has3-deficient mice was decreased 28 days after ligation, and neointimal hyperplasia was strongly inhibited. However, medial and luminal areas were unaffected. Cell density, proliferation, and apoptosis were not altered, suggesting a proportional decrease of both, the number of cells and extracellular matrix. In addition, endothelial function as determined by acetylcholine-induced relaxation of aortic rings, immunoblotting of endothelial nitric oxide synthase, and arterial blood pressure were not affected. Furthermore, the oxidative stress response was not affected as determined in total protein extracts from aortae. Transcriptome analysis comparing control versus ligated carotid arteries hinted toward a mitigated differential regulation of various signaling pathways in Has3-deficient mice in response to ligation that were related to vascular smooth muscle cell (VSMC) migration, including focal adhesions, integrins, mitogen-activated protein kinase, and phosphatidylinositol signaling system. Lentiviral overexpression of HAS3 in VSMC supported the migratory phenotype of VSMC in response to platelet-derived growth factor BB in vitro. Accordingly, knockdown of HAS3 reduced the migratory response to platelet-derived growth factor BB and in addition decreased the expression of PDGF-B mRNA. CONCLUSIONS: HAS3-mediated HA synthesis after vessel injury supports seminal signaling pathways in activation of VSMC, increases platelet-derived growth factor BB-mediated migration, and in turn enhances neointimal hyperplasia in vivo.

Increased cathepsin D protein expression is a biomarker for osteosarcomas, pulmonary metastases and other bone malignancies.

Cancer proteomics provide a powerful approach to identify biomarkers for personalized medicine. Particularly, biomarkers for early detection, prognosis and therapeutic intervention of bone cancers, especially osteosarcomas, are missing. Initially, we compared two-dimensional gel electrophoresis (2-DE)-based protein expression pattern between cell lines of fetal osteoblasts, osteosarcoma and pulmonary metastasis derived from osteosarcoma. Two independent statistical analyses by means of PDQuest® and SameSpot® software revealed a common set of 34 differentially expressed protein spots (p < 0.05). 17 Proteins were identified by mass spectrometry and subjected to Ingenuity Pathway Analysis resulting in one high-ranked network associated with Gene Expression, Cell Death and Cell-To-Cell Signaling and Interaction. Ran/TC4-binding protein (RANBP1) and Cathepsin D (CTSD) were further validated by Western Blot in cell lines while the latter one showed higher expression differences also in cytospins and in clinical samples using tissue microarrays comprising osteosarcomas, metastases, other bone malignancies, and control tissues. The results show that protein expression patterns distinguish fetal osteoblasts from osteosarcomas, pulmonary metastases, and other bone diseases with relevant sensitivities between 55.56% and 100% at ≥87.50% specificity. Particularly, CTSD was validated in clinical material and could thus serve as a new biomarker for bone malignancies and potentially guide individualized treatment regimes.