<p>The miR-371~373/TGFBR2 downstream target ID1 regulates tumor growth and metastatic dissemination potential</p>

<p>Investigation of SOX2 and BMP signaling as potential miR-371~373/TGFBR2 targets</p>

Background: CCN1/CYR61 is a matricellular protein belonging to CCN protein family that contains six secreted proteins associated with extracellular matrix signaling [1]. It acts as an enhancer of cutaneous wound healing process by preventing hypertrophic scar formation through induction of myofibroblast (MFB) senescence [2]. In liver fibrosis, senescent cells are primarily derived from activated hepatic stellate cells (HSC) that initially proliferate in response to liver damage and transdifferentiate into MFB. Activated HSC and transdifferentiated MFB are one major source of CCN1 [3]. Methods: We tested if CCN1 act as a senescence inducer to attenuate liver fibrogenesis by means of adenoviral CCN1 gene transfer in primary HSC, MFB, CFSC-2G, and LX-2. Results: The overwhelming concentrations of CCN1 protein resulted in an overload of the endoplasmic reticulum (ER) and compensatory unfolded protein response (UPR) in all these cells. The UPR resulted in upregulation of ER chaperones (BIP/Grp78, Grp94) and leads to an activation of IRE1α as evidenced by spliced XBP1 mRNA with IRE1α-induced JNK phosphorylation. The UPR arm PERK and eIF2a was phosphorylated, and CHOP upregulated. Ad5-CMV-CCN1 induced HSC apoptosis by proteolytic cleavage of caspase-12, caspase-9, and caspase-3 resulting in positive TUNEL stain. Remarkably, CCN1 effectively blocked collagen type I expression at both mRNA and protein levels. Conclusions: High concentrations of the matricellular protein CCN1 induce HSC apoptosis through ER stress and UPR. Therapeutic CCN1 gene transfer might be useful to mitigate liver fibrosis when cell-specific targeted to HSC and MFB.

<p>Short hairpin (sh) RNA knockdown control of TGFBR2</p>

CCN1/CYR61 is a matricellular protein of the CCN family, comprising six secreted proteins specifically associated with the extracellular matrix (ECM). CCN1 acts as an enhancer of the cutaneous wound healing process by preventing hypertrophic scar formation through induction of myofibroblast senescence. In liver fibrosis, the senescent cells are primarily derived from activated hepatic stellate cells (HSC) that initially proliferate in response to liver damage and are the major source of ECM. We investigate here the possible use of CCN1 as a senescence inducer to attenuate liver fibrogenesis by means of adenoviral gene transfer in primary HSC, myofibroblasts (MFB) and immortalized HSC lines (i.e. LX-2, CFSC-2G). Infection with Ad5-CMV-CCN1 induced large amounts of CCN1 protein in all these cells, resulting in an overload of the endoplasmic reticulum (ER) and in a compensatory unfolded protein response (UPR). The UPR resulted in upregulation of ER chaperones including BIP/Grp78, Grp94 and led to an activation of IRE1α as evidenced by spliced XBP1 mRNA with IRE1α-induced JNK phosphorylation. The UPR arm PERK and eIF2a was phosphorylated, combined with significant CHOP upregulation. Ad5-CMV-CCN1 induced HSC apoptosis that was evident by proteolytic cleavage of caspase-12, caspase-9 and the executor caspase-3 and positive TUNEL stain. Remarkably, Ad5-CMV-CCN1 effectively reduced collagen type I mRNA expression and protein. We conclude that the matricellular protein CCN1 gene transfer induces HSC apoptosis through ER stress and UPR.

<p>Additional colony formation assays and size measurement of 2D colonies and 3D spheroids</p>

Cell plasticity of ITIH5-expressing MDA-MB-231 single-cell clones. This figure shows morphological characteristics of independent MDA-MB-231 single-cell clones using phase-contrast microscopy. (DOCX 109Â kb)

The cultivation of cells in a favorable artificial environment has become a versatile tool in cellular and molecular biology. Cultured primary cells and continuous cell lines are indispensable in investigations of basic, biomedical, and translation research. However, despite their important role, cell lines are frequently misidentified or contaminated by other cells, bacteria, fungi, yeast, viruses, or chemicals. In addition, handling and manipulating of cells is associated with specific biological and chemical hazards requiring special safeguards such as biosafety cabinets, enclosed containers, and other specialized protective equipment to minimize the risk of exposure to hazardous materials and to guarantee aseptic work conditions. This review provides a brief introduction about the most common problems encountered in cell culture laboratories and some guidelines on preventing or tackling respective problems.

Sepsis is a major health burden with insufficiently understood mechanisms of inflammation and immune paralysis, leading to a life-threatening critical illness. The secreted frizzled related protein 5 (SFRP5) acts as an anti-inflammatory adipokine by antagonizing the Wnt5a pathway. The aim of this study was to elucidate the role of SFRP5 in critical illness and sepsis and to determine its value as a prognostic biomarker for mortality. We analyzed SFRP5 serum concentrations of 223 critically ill patients at admission to a medical intensive care unit (ICU) and compared those to 24 healthy individuals. SFRP5 serum concentrations were significantly decreased in critical illness as compared to healthy controls (24.66 vs. 100 ng/mL, p = 0.029). Even lower serum concentrations were found in septic as compared to nonseptic critically ill patients (19.21 vs. 32.83 ng/mL, p = 0.031). SFRP5 concentrations correlated with liver disease, age, anti-inflammation, and metabolic parameters. Furthermore, patients with sepsis recovered levels of SFRP5 in the first week of ICU treatment. SFRP5 levels at admission predicted short-term mortality in critically ill but not in septic patients. This study points to the role of the anti-inflammatory mediator SFRP5 not only in sepsis but also in nonseptic critically ill patients and associates high levels of SFRP5 to worse outcomes, predominantly in nonseptic critically ill patients.

The liver is the most central organ and the largest gland of the body that influences and controls a variety of metabolic and catabolic processes. It produces inconceivable many essential proteins, is responsible for the recovery of various food components, degrades toxins, mediates the bile production, and is involved in the excretion of unwanted metabolites. Several of these anabolic or catabolic functions of the liver depend on trace elements. These are either integral part of enzymes, cofactors, or act as chemical catalysts. Therefore, a lack of trace elements can lead to organ failure or systemic illness. Conversely, excessive hepatic trace element deposition resulting from genetic disorders, intoxication, extensive dietary supply, or long-term parenteral nutrition may cause hepatic inflammation, fibrosis, cirrhosis, and even hepatocellular carcinoma. Although specific serum parameters currently allow rough assessment of metal deficit and excess, the precise quantification of hepatic metal content in liver is presently only possible by different titration or staining techniques of biopsy specimens. Recently, novel innovative metal imaging techniques were developed that are on the way to replace these traditional methods. In the present review, we summarize the function of different trace elements in liver health and disease and discuss the present knowledge on how quantitative biometal imaging techniques such as synchrotron X-ray fluorescence microscopy, secondary ion mass spectrometry, and laser ablation inductively coupled plasma mass spectrometry enrich diagnostics in the detection and quantification of hepatic metal disorders. We will further discuss sample preparation, sensitivity, spatial resolution, specificity, quantification strategies, and potential future applications of metal bioimaging in experimental research and clinical daily routine. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 35:666-686, 2016.

The induction of experimental obstructive cholestasis is a reliable model for cholestatic liver diseases in rodents. Bile duct ligation (BDL) in mice provokes typical time-dependent morphological and structural changes in the liver, ranging from liver cell injury and elevated serum enzyme levels after several days, to a severe inflammatory response in the liver after 5-7 days, up to an advanced hepatic fibrosis as soon as three to four weeks after surgical ligation of the common biliary duct. Upon BDL induction, hepatic stellate cells become activated and transdifferentiate into myofibroblasts that produce extracellular matrix proteins such as collagen. In principle, the periportal fibrosis induced by BDL in rat livers is reversible. After the relief of a biliary obstruction, the liver has the capacity to revert to a nearly normal histological architecture and a fully normal biochemical function. When BDL surgery is performed by an experienced scientist, this model has very high reproducibility among all fibrotic models. All these factors corroborate the outstanding value of this model for basic and translational research in biomedicine and hepatology. Nevertheless, this model can result in significant variations when surgery is carried out by untrained personnel or when unconscious modifications are implemented that affect the quality of the intervention. A detailed protocol is provided here for the provision of reliable and reproducible BDL in mice.