Signal intensity in the dentate nucleus after cumulative dose of Gd-EOB-DTPA: First results of a prospective longitudinal study.

Gadolinium ethoxybenzyl-diethylenetriaminepentaacetic acid (Gd-EOB-DTPA) is a hepatocyte-specific, linear ionic contrast agent for MRI. In comparison to other linear contrast agents Gd-EOB-DTPA is excreted equally through liver and kidneys. This prospective longitudinal study investigates the signal intensity (SI) in the dentate nucleus (DN) on unenhanced T1-weighted images after repetitive application of Gd-EOB-DTPA. 46 patients were included into the study and 107 MRI examinations were performed. Statistical analysis of 25 patients showed no significant correlation between cumulative dose of Gd-EOB-DTPA and SI change and between the DN/Pons ratiolast and the mean DN/Pons ratiofirst. Subgroup analysis however revealed a significant correlation for one out of two readers. Gd-EOB-DTPA deposition could not be proven in the framework of this study.

Long Noncoding RNA TYKRIL Plays a Role in Pulmonary Hypertension via the p53-mediated Regulation of PDGFRß.

Rationale: Long noncoding RNAs (lncRNAs) are emerging as important regulators of diverse biological functions. Their role in pulmonary arterial hypertension (PAH) remains to be explored.Objectives: To elucidate the role of TYKRIL (tyrosine kinase receptor-inducing lncRNA) as a regulator of p53/ PDGFRß (platelet-derived growth factor receptor ß) signaling pathway and to investigate its role in PAH.Methods: Pericytes and pulmonary arterial smooth muscle cells exposed to hypoxia and derived from patients with idiopathic PAH were analyzed with RNA sequencing. TYKRIL knockdown was performed in above-mentioned human primary cells and in precision-cut lung slices derived from patients with PAH.Measurements and Main Results: Using RNA sequencing data, TYKRIL was identified to be consistently upregulated in pericytes and pulmonary arterial smooth muscles cells exposed to hypoxia and derived from patients with idiopathic PAH. TYKRIL knockdown reversed the proproliferative (n = 3) and antiapoptotic (n = 3) phenotype induced under hypoxic and idiopathic PAH conditions. Owing to the poor species conservation of TYKRIL, ex vivo studies were performed in precision-cut lung slices from patients with PAH. Knockdown of TYKRIL in precision-cut lung slices decreased the vascular remodeling (n = 5). The number of proliferating cell nuclear antigen-positive cells in the vessels was decreased and the number of terminal deoxynucleotide transferase-mediated dUTP nick end label-positive cells in the vessels was increased in the LNA (locked nucleic acid)-treated group compared with control. Expression of PDGFRß, a key player in PAH, was found to strongly correlate with TYKRIL expression in the patient samples (n = 12), and TYKRIL knockdown decreased PDGFRß expression (n = 3). From the transcription factor-screening array, it was observed that TYKRIL knockdown increased the p53 activity, a known repressor of PDGFRß. RNA immunoprecipitation using various p53 mutants demonstrated that TYKRIL binds to the N-terminal of p53 (an important region for p300 interaction with p53). The proximity ligation assay revealed that TYKRIL interferes with the p53-p300 interaction (n = 3) and regulates p53 nuclear translocation.Conclusions: TYKRIL plays an important role in PAH by regulating the p53/PDGFRß axis.

Hematopoietic Deficiency of the Long Noncoding RNA MALAT1 Promotes Atherosclerosis and Plaque Inflammation.

BACKGROUND: The majority of the human genome comprises noncoding sequences, which are in part transcribed as long noncoding RNAs (lncRNAs). lncRNAs exhibit multiple functions, including the epigenetic control of gene expression. In this study, the effect of the lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) on atherosclerosis was examined. METHODS: The effect of MALAT1 on atherosclerosis was determined in apolipoprotein E-deficient (Apoe-/-) MALAT1-deficient (Malat1-/-) mice that were fed with a high-fat diet and by studying the regulation of MALAT1 in human plaques. RESULTS: Apoe-/- Malat1-/- mice that were fed a high-fat diet showed increased plaque size and infiltration of inflammatory CD45+ cells compared with Apoe-/- Malat1+/+ control mice. Bone marrow transplantation of Apoe-/- Malat1-/- bone marrow cells in Apoe-/- Malat1+/+ mice enhanced atherosclerotic lesion formation, which suggests that hematopoietic cells mediate the proatherosclerotic phenotype. Indeed, bone marrow cells isolated from Malat1-/- mice showed increased adhesion to endothelial cells and elevated levels of proinflammatory mediators. Moreover, myeloid cells of Malat1-/- mice displayed enhanced adhesion to atherosclerotic arteries in vivo. The anti-inflammatory effects of MALAT1 were attributed in part to reduction of the microRNA miR-503. MALAT1 expression was further significantly decreased in human plaques compared with normal arteries and was lower in symptomatic versus asymptomatic patients. Lower levels of MALAT1 in human plaques were associated with a worse prognosis. CONCLUSIONS: Reduced levels of MALAT1 augment atherosclerotic lesion formation in mice and are associated with human atherosclerotic disease. The proatherosclerotic effects observed in Malat1-/- mice were mainly caused by enhanced accumulation of hematopoietic cells.

Safety and efficacy of transarterial chemoembolization with degradable starch microspheres (DSM-TACE) in the treatment of secondary liver malignancies.

PURPOSE: To evaluate the safety and efficacy of degradable starch microspheres (DSM) as embolic agents in transarterial chemoembolization (TACE) in the treatment of secondary liver metastases. METHODS: This was a national, multicenter observational study. Primary endpoints were safety and treatment response according to Modified Response Evaluation Criteria in Solid Tumors (mRECIST) criteria. RESULTS: A total of 77 DSM-TACE procedures were performed in 20 patients. Minor immediate adverse events (AEs) were epigastric pain with an incidence of 45.5% (35/77), and nausea and vomiting at an incidence of 23.4% (18/77). Delayed minor AEs were epigastric pain in 13/77 (16.9%) treatments and nausea and vomiting in 10 (13.0%) treatments. No severe AEs were documented. Therapeutic efficacy of DSM-TACE procedures according to mRECIST was as follows: complete response 0/77, partial response 17/77, stable disease 33/77 and progressive disease 6/77, no data was available for 21/77 treatments. Overall, objective response was achieved in 8 of 20 patients (40.0%). CONCLUSION: DSM as embolic agent for TACE is safe in the treatment of liver metastases. An objective response in 40.0% of patients and disease control in 64.9% of procedures was achieved, and this should lead to further evaluation of DSM-TACE as treatment option for nonresectable liver metastases.

Characterization of Focal Liver Lesions using CEUS and MRI with Liver-Specific Contrast Media: Experience of a Single Radiologic Center.

AIM: The purpose of this study was to compare contrast-enhanced ultrasound (CEUS), magnetic resonance imaging (MRI) using liver-specific contrast agent and a combination of both for the characterization of focal liver lesions (FLL). METHODS: 83 patients with both benign and malignant liver lesions were examined using CEUS and MRI after the intravenous administration of liver-specific contrast media. All patients had inconclusive results from prior imaging examinations. Histopathological specimens could be obtained in 53 patients. Ultrasound was performed using a multi-frequency curved probe (1 - 6 MHz) after the injection of 1 - 2.4 ml ultrasound contrast media. The sensitivity, specificity, positive predictive value and negative predictive value of CEUS, MRI and a combination of both (CEUS + MRI) were compared. RESULTS: The sensitivity, specificity, positive and negative predictive values regarding lesion classification were 90.9 %, 70.6 %, 92.3 % and 66.6 %, respectively, for CEUS; 90.9 %, 82.4 %, 95.2 % and 70.0 %, respectively, for MRI; and 96.9 %, 70.6 %, 92.7 % and 85.7 % respectively, for CEUS + MRI. There were no statistically significant differences. 6 malignant lesions were missed using CEUS or MRI alone (false negatives). The use of both modalities combined reduced the false-negative results to 2. CONCLUSION: CEUS and MRI with liver-specific contrast media are very reliable and of equal informative value in the characterization of focal liver lesions. The number of false-negative results can be decreased using a combination of the two methods.

Transarterial chemoembolization (TACE) with degradable starch microspheres (DSM) in hepatocellular carcinoma (HCC): multi-center results on safety and efficacy.

BACKGROUND: Hepatocellular carcinoma (HCC) is the 3rd leading cause of cancer-related death worldwide. The majority of HCCs are diagnosed in a stage that is not eligible for curative resection. For intermediate stage HCC, transarterial chemoembolization (TACE) is the recommended treatment. We evaluated the safety and efficacy of DSM (degradable starch microspheres) as embolic agent in transarterial chemoembolization (TACE) for the treatment of intermediate stage, non-resectable hepatocellular carcinoma (HCC). METHODS AND FINDINGS: A national, multi-center observational study on the safety and efficacy of DSM-TACE for the treatment of intermediate HCC was conducted. The recruitment period for the study was from January 2010 to June 2014. The primary endpoints were safety and treatment response according to the mRECIST criteria. A total of 179 DSM-TACE procedures in 50 patients were included in the analysis. The therapeutic efficacy assessed with mRECIST was as follows: complete response (n=1; 2 %), 21 partial response (42 %), 13 stable disease (26 %), 9 progressive disease (18 %), and 6 incomplete data (12 %). Thus, the objective response rate was 44% (n=22) and disease control rate was 70% (n=35). A total of 76 immediate adverse events (AE) and 2 severe adverse events (SAE) were recorded. Forty-eight percent of patients (n=24) did not encounter any immediate AE/SAE. Between treatments, a total of 66 AE and one SAE were recorded. Twenty-four patients (48 %) did not encounter any AE/SAE in between treatments. CONCLUSION: The use of DSM as a TACE embolic agent appears to be safe for the treatment of HCC and has promising efficacy.

Identification and Functional Characterization of Hypoxia-Induced Endoplasmic Reticulum Stress Regulating lncRNA (HypERlnc) in Pericytes.

RATIONALE: Pericytes are essential for vessel maturation and endothelial barrier function. Long noncoding RNAs regulate many cellular functions, but their role in pericyte biology remains unexplored. OBJECTIVE: Here, we investigate the effect of hypoxia-induced endoplasmic reticulum stress regulating long noncoding RNAs (HypERlnc, also known as ENSG00000262454) on pericyte function in vitro and its regulation in human heart failure and idiopathic pulmonary arterial hypertension. METHODS AND RESULTS: RNA sequencing in human primary pericytes identified hypoxia-regulated long noncoding RNAs, including HypERlnc. Silencing of HypERlnc decreased cell viability and proliferation and resulted in pericyte dedifferentiation, which went along with increased endothelial permeability in cocultures consisting of human primary pericyte and human coronary microvascular endothelial cells. Consistently, Cas9-based transcriptional activation of HypERlnc was associated with increased expression of pericyte marker genes. Moreover, HypERlnc knockdown reduced endothelial-pericyte recruitment in Matrigel assays (P<0.05). Mechanistically, transcription factor reporter arrays demonstrated that endoplasmic reticulum stress-related transcription factors were prominently activated by HypERlnc knockdown, which was confirmed via immunoblotting for the endoplasmic reticulum stress markers IRE1α (P<0.001), ATF6 (P<0.01), and soluble BiP (P<0.001). Kyoto encyclopedia of genes and gene ontology pathway analyses of RNA sequencing experiments after HypERlnc knockdown indicate a role in cardiovascular disease states. Indeed, HypERlnc expression was significantly reduced in human cardiac tissue from patients with heart failure (P<0.05; n=19) compared with controls. In addition, HypERlnc expression significantly correlated with pericyte markers in human lungs derived from patients diagnosed with idiopathic pulmonary arterial hypertension and from donor lungs (n=14). CONCLUSIONS: Here, we show that HypERlnc regulates human pericyte function and the endoplasmic reticulum stress response. In addition, RNA sequencing analyses in conjunction with reduced expression of HypERlnc in heart failure and correlation with pericyte markers in idiopathic pulmonary arterial hypertension indicate a role of HypERlnc in human cardiopulmonary disease.

Degradable Starch Microspheres Transcatheter Arterial Chemoembolization (DSM-TACE) in Intrahepatic Cholangiocellular Carcinoma (ICC): Results from a National Multi-Center Study on Safety and Efficacy.

BACKGROUND The aim of this study was to evaluate the safety and efficacy of DSM (degradable starch microspheres) as an embolic agent in transarterial chemoembolization in the treatment of intrahepatic cholangiocellular carcinoma (ICC). MATERIAL AND METHODS This was a national, multi-center observational cohort study on the safety and efficacy of DSM-TACE using mitomycin, gemcitabine, cisplatin, doxorubicin, and carboplatin in palliative treatment of ICC. Recruitment period for the study was from January 2010 to June 2014. Primary endpoints were toxicity, safety, and response according to mRECIST criteria. RESULTS Twenty-five DSM-TACE procedures in cases of advanced ICC were performed in seven patients. Nausea and vomiting occurred as adverse event (AE) in eight out of 25 treatments (32%), with seven of eight events (87.5%) associated with the use of gemcitabine. In 11 out of 25 treatments (44%) moderate, transient epigastric pain was registered as an adverse event (AE) within 24 hours of DSM-TACE. One case (1/25) of severe AE (4%) with thrombocytopenia led to discontinuation of the DSM-TACE-treatment. A total of 25 DSM-TACE procedures with complete clinical and imaging follow-up over a two-year-period were analyzed: objective response (OR) was achieved in three of 25 treatments (12%) Disease control (DC) was achieved in 44% (11/25) of treatments; progress was registered in 4% (1/25). CONCLUSIONS The use of DSM as an embolic agent for TACE is safe in the treatment of ICC. A standardized anti-emetic medication should be established, especially when using gemcitabine. Further prospective studies need to be conducted to find the most suitable, standardized DSM-TACE treatment regime.

Evaluation of a Robotic Assistance-System For Percutaneous Computed Tomography-Guided (CT-Guided) Facet Joint Injection: A Phantom Study.

BACKGROUND The aim of this study was to compare robotic assisted and freehand facet joint puncture on a phantom model in regards to time requirements and puncture accuracy. MATERIAL AND METHODS Forty facet joints were punctured, 20 using a robotic guidance system and 20 using a freehand procedure. Side and height of the facet joints were randomized and identical for both groups. Procedural accuracy, defined as axial and sagittal deviation, as well as the number of corrections were assessed. Procedure times for each step were documented and time requirements for pre-positioning, reconstruction, planning, and total intervention were calculated. RESULTS Total procedure time for robotic guidance was 259±111 seconds versus 119±77 seconds for freehand procedure (p=1.0). Procedural accuracy for robotic guidance was significantly higher with 0 corrections versus 1.3 corrections for freehand procedure (p=0.02). Needle deviation in the robotics arm was 0.35±1.1 mm in the axial and 2.15±1.2 mm in the sagittal reconstruction. CONCLUSIONS Robotic assisted puncture of the facet joint allowed accurate positioning of the needle with a lower number of needle readjustments. Higher procedural accuracy was marginally offset by a slightly longer intervention time.

Laminar shear stress inhibits endothelial cell metabolism via KLF2-mediated repression of PFKFB3.

OBJECTIVE: Cellular metabolism was recently shown to regulate endothelial cell phenotype profoundly. Whether the atheroprotective biomechanical stimulus elicited by laminar shear stress modulates endothelial cell metabolism is not known. APPROACH AND RESULTS: Here, we show that laminar flow exposure reduced glucose uptake and mitochondrial content in endothelium. Shear stress-mediated reduction of endothelial metabolism was reversed by silencing the flow-sensitive transcription factor Krüppel-like factor 2 (KLF2). Endothelial-specific deletion of KLF2 in mice induced glucose uptake in endothelial cells of perfused hearts. KLF2 overexpression recapitulates the inhibitory effects on endothelial glycolysis elicited by laminar flow, as measured by Seahorse flux analysis and glucose uptake measurements. RNA sequencing showed that shear stress reduced the expression of key glycolytic enzymes, such as 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 (PFKFB3), phosphofructokinase-1, and hexokinase 2 in a KLF2-dependent manner. Moreover, KLF2 represses PFKFB3 promoter activity. PFKFB3 knockdown reduced glycolysis, and overexpression increased glycolysis and partially reversed the KLF2-mediated reduction in glycolysis. Furthermore, PFKFB3 overexpression reversed KLF2-mediated reduction in angiogenic sprouting and network formation. CONCLUSIONS: Our data demonstrate that shear stress-mediated repression of endothelial cell metabolism via KLF2 and PFKFB3 controls endothelial cell phenotype.

Long noncoding RNA MALAT1 regulates endothelial cell function and vessel growth.

RATIONALE: The human genome harbors a large number of sequences encoding for RNAs that are not translated but control cellular functions by distinct mechanisms. The expression and function of the longer transcripts namely the long noncoding RNAs in the vasculature are largely unknown. OBJECTIVE: Here, we characterized the expression of long noncoding RNAs in human endothelial cells and elucidated the function of the highly expressed metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). METHODS AND RESULTS: Endothelial cells of different origin express relative high levels of the conserved long noncoding RNAs MALAT1, taurine upregulated gene 1 (TUG1), maternally expressed 3 (MEG3), linc00657, and linc00493. MALAT1 was significantly increased by hypoxia and controls a phenotypic switch in endothelial cells. Silencing of MALAT1 by small interfering RNAs or GapmeRs induced a promigratory response and increased basal sprouting and migration, whereas proliferation of endothelial cells was inhibited. When angiogenesis was further stimulated by vascular endothelial growth factor, MALAT1 small interfering RNAs induced discontinuous sprouts indicative of defective proliferation of stalk cells. In vivo studies confirmed that genetic ablation of MALAT1 inhibited proliferation of endothelial cells and reduced neonatal retina vascularization. Pharmacological inhibition of MALAT1 by GapmeRs reduced blood flow recovery and capillary density after hindlimb ischemia. Gene expression profiling followed by confirmatory quantitative reverse transcriptase-polymerase chain reaction demonstrated that silencing of MALAT1 impaired the expression of various cell cycle regulators. CONCLUSIONS: Silencing of MALAT1 tips the balance from a proliferative to a migratory endothelial cell phenotype in vitro, and its genetic deletion or pharmacological inhibition reduces vascular growth in vivo.

Drosophila miR-277 controls branched-chain amino acid catabolism and affects lifespan.

Development, growth and adult survival are coordinated with available metabolic resources, ascertaining that the organism responds appropriately to environmental conditions. MicroRNAs are short (21-23 nt) regulatory RNAs that confer specificity on the RNA-induced silencing complex (RISC) to inhibit a given set of mRNA targets. We profiled changes in miRNA expression during adult life in Drosophila melanogaster and determined that miR-277 is downregulated during adult life. Molecular analysis revealed that this miRNA controls branched-chain amino acid (BCAA) catabolism and as a result it can modulate the activity of the TOR kinase, a central growth regulator, in cultured cells. Metabolite analysis in cultured cells as well as flies suggests that the mechanistic basis may be an accumulation of branched-chain α-keto-acids (BCKA), rather than BCAAs, thus avoiding potentially detrimental consequences of increased branched chain amino acid levels on e.g., translational fidelity. Constitutive miR-277 expression shortens lifespan and is synthetically lethal with reduced insulin signaling, indicating that metabolic control underlies this phenotype. Transgenic inhibition with a miRNA sponge construct also shortens lifespan, in particular on protein-rich food. Thus, optimal metabolic adaptation appears to require tuning of cellular BCAA catabolism by miR-277.

Curcumin inhibits prostate cancer metastasis in vivo by targeting the inflammatory cytokines CXCL1 and -2.

In America and Western Europe, prostate cancer is the second leading cause of death in men. Emerging evidence suggests that chronic inflammation is a major risk factor for the development and metastatic progression of prostate cancer. We previously reported that the chemopreventive polyphenol curcumin inhibits the expression of the proinflammatory cytokines CXCL1 and -2 leading to diminished formation of breast cancer metastases. In this study, we analyze the effects of curcumin on prostate carcinoma growth, apoptosis and metastasis. We show that curcumin inhibits translocation of NFκB to the nucleus through the inhibition of the IκB-kinase (IKKß, leading to stabilization of the inhibitor of NFκB, IκBα, in PC-3 prostate carcinoma cells. Inhibition of NFκB activity reduces expression of CXCL1 and -2 and abolishes the autocrine/paracrine loop that links the two chemokines to NFκB. The combination of curcumin with the synthetic IKKß inhibitor, SC-541, shows no additive or synergistic effects indicating that the two compounds share the target. Treatment of the cells with curcumin and siRNA-based knockdown of CXCL1 and -2 induce apoptosis, inhibit proliferation and downregulate several important metastasis-promoting factors like COX2, SPARC and EFEMP. In an orthotopic mouse model of hematogenous metastasis, treatment with curcumin inhibits statistically significantly formation of lung metastases. In conclusion, chronic inflammation can induce a metastasis prone phenotype in prostate cancer cells by maintaining a positive proinflammatory and prometastatic feedback loop between NFκB and CXCL1/-2. Curcumin disrupts this feedback loop by the inhibition of NFκB signaling leading to reduced metastasis formation in vivo.

A small RNA response at DNA ends in Drosophila.

Small RNAs have been implicated in numerous cellular processes, including effects on chromatin structure and the repression of transposons. We describe the generation of a small RNA response at DNA ends in Drosophila that is analogous to the recently reported double-strand break (DSB)-induced RNAs or Dicer- and Drosha-dependent small RNAs in Arabidopsis and vertebrates. Active transcription in the vicinity of the break amplifies this small RNA response, demonstrating that the normal messenger RNA contributes to the endogenous small interfering RNAs precursor. The double-stranded RNA precursor forms with an antisense transcript that initiates at the DNA break. Breaks are thus sites of transcription initiation, a novel aspect of the cellular DSB response. This response is specific to a double-strand break since nicked DNA structures do not trigger small RNA production. The small RNAs are generated independently of the exact end structure (blunt, 3'- or 5'-overhang), can repress homologous sequences in trans and may therefore--in addition to putative roles in repair--exert a quality control function by clearing potentially truncated messages from genes in the vicinity of the break.