Liver fibrosis is orchestrated by a complex network of signaling pathways regulating the deposition of extracellular matrix proteins during fibrogenesis. MicroRNAs (miRNAs) represent a family of small noncoding RNAs controlling translation and transcription of many genes. Recently, miRNAs have been suggested to crucially modulate cellular processes in the liver such as hepatocarcinogenesis. However, their role in liver fibrosis is not well understood. We systematically analyzed the regulation of miRNAs in a mouse model of carbon tetrachloride-induced hepatic fibrogenesis (CCl 4) by gene array analysis, which revealed a panel of miRNA that were specifically regulated in livers of mice undergoing hepatic fibrosis. Within those, all three members of the miR-29-family were significantly down-regulated in livers of CCl 4-treated mice as well as in mice that underwent bile duct ligation. Specific regulation of miR-29 members in murine fibrosis models correlated with lower expression of miR-29 in livers from patients with advanced liver fibrosis. Moreover, patients with advanced liver cirrhosis showed significantly lower levels of miR-29a in their serum when compared with healthy controls or patients with early fibrosis. On a cellular level, down-regulation of miR-29 in murine hepatic stellate cells (HSCs) was mediated by transforming growth factor beta (TGF-b) as well as inflammatory signals, namely, lipopolysaccharide (LPS) and nuclear factor kappa B (NF-jB). Furthermore, overexpression of miR-29b in murine HSC resulted in down-regulation of collagen expression. Conclusion: Our data indicate that miR-29 mediates the regulation of liver fibrosis and is part of a signaling nexus involving TGF-band NF-jB-dependent down-regulation of miR-29 family members in HSC with subsequent up-regulation of extracellular matrix genes. Thus they may represent targets for novel therapeutic strategies against hepatic fibrogenesis and also might evolve as biomarkers in the diagnosis of liver fibrosis.

The MAP3-kinase TGF-beta-activated kinase 1 (TAK1) critically modulates innate and adaptive immune responses and connects cytokine stimulation with activation of inflammatory signaling pathways. Here, we report that conditional ablation of TAK1 in liver parenchymal cells (hepatocytes and cholangiocytes) causes hepatocyte dysplasia and early-onset hepatocarcinogenesis, coinciding with biliary ductopenia and cholestasis. TAK1-mediated cancer suppression is exerted through activating NF-kappaB in response to tumor necrosis factor (TNF) and through preventing Caspase-3-dependent hepatocyte and cholangiocyte apoptosis. Moreover, TAK1 suppresses a procarcinogenic and pronecrotic pathway, which depends on NF-kappaB-independent functions of the I kappaB-kinase (IKK)-subunit NF-kappaB essential modulator (NEMO). Therefore, TAK1 serves as a gatekeeper for a protumorigenic, NF-kappaB-independent function of NEMO in parenchymal liver cells.

MicroRNA (miRNA) levels in serum have recently emerged as potential novel biomarkers for various diseases. miRNAs are routinely measured by standard quantitative real-time PCR (qPCR); however, the high sensitivity of qPCR demands appropriate normalization to correct for nonbiological variation. Presently, RNU6B (U6) is used for data normalization of circulating miRNAs in many studies. However, it was suggested that serum levels of U6 themselves might differ between individuals. Therefore, no consensus has been reached on the best normalization strategy in ‘circulating miRNA'. We analyzed U6 levels as well as levels of spiked-in SV40-RNA in sera of 44 healthy volunteers, 203 intensive care unit patients and 64 patients with liver fibrosis. Levels of U6 demonstrated a high variability in sera of healthy donors, patients with critical illness and liver fibrosis. This high variability could also be confirmed in sera of mice after the cecal ligation and puncture procedure. Most importantly, levels of circulating U6 were significantly upregulated in sera of patients with critical illness and sepsis compared with controls and correlated with established markers of inflammation. In patients with liver fibrosis, U6 levels were significantly downregulated. In contrast, levels of spiked-in SV40 displayed a significantly higher stability both in human cohorts (healthy, critical illness, liver fibrosis) and in mice. Thus, we conclude that U6 levels in the serum are dysregulated in a disease-specific manner. Therefore, U6 should not be used for data normalization of circulating miRNAs in inflammatory diseases and previous studies using this approach should be interpreted with caution. Further studies are warranted to identify specific regulatory processes of U6 levels in sepsis and liver fibrosis.

The accurate assessment of the presence and extent of vascular disease, and planning of vascular interventions based on MRA requires the determination of vessel dimensions. The current standard is based on measuring vessel diameters on maximum intensity projections (MIPs) using calipers. In order to increase the accuracy and reproducibility of the method, automated analysis of the 3D MR data is required. A novel method for automatically determining the trajectory of the vessel of interest, the luminal boundaries, and subsequent the vessel dimensions is presented. The automated segmentation in 3D uses deformable models, combined with knowledge of the acquisition protocol. The trajectory determination was tested on 20 in vivo studies of the abdomen and legs. In 93% the detected trajectory followed the vessel. Determination of the vessel morphology along a vessel segment is important in grading the presence and extent of possible vascular stenoses. Until recently, most examinations of vascular stenoses were carried out using x-ray angiography (XA). This technology has been regarded as the gold standard in the evaluation of stenoses. However, several problems are associated with this imaging modality. First, since XA is a projection technique, overprojection of vessels can occur even if the view angle is set optimally. Second, an ionizing nephrotoxic contrast agent has to be administered by means of a catheter, a technique that is associated with a definite (although relatively small) morbidity and mortality risk. Finally, the patient and the personnel of the catheterization laboratory are exposed to x-ray radiation.Magnetic resonance angiography (MRA), on the other hand, is a technique that produces three-dimensional (3D) images. Consequently, there is no risk of overprojection of vessels. MRA can be applied without the use of a contrast agent, although the signal-to-noise ratio (SNR) increases if a contrast agent is used. This contrast agent is normally administered intravenously, which presents minimal risks to the patient (1,2).MRA data sets are generally evaluated on 2D maximum intensity projections (MIP); however, it is known that this leads to underestimation of the vessel width, and a decreased SNR. The interpretation is carried out by either visual inspection or by caliper measurements (3-7).To improve the conventional analysis of MRA, it would be desirable to obtain quantitative morphological information directly from the 3D images and not from the projections. To accomplish this, accurate 3D segmentation tools are required.Vessel segmentation of 3D images has been investigated by many researchers (8 -16). However, the majority of this research focused on enhancing the 3D visualization of the vascular structures in the image, and not on accurate quantification of these structures.In this work a novel approach for quantitative vessel analysis of MRA images is introduced and validated. The approach uses knowledge about the image acquisition procedure to accurately determine the vessel boundaries. The techniq...

This paper presents new approaches for the assessment of the arterial and reference diameters in (cardio-)vascular X-ray images, designed to overcome the problems experienced in conventional quantitative coronary and vascular angiography approaches. In single or “straight” vessel segments, the arterial and reference diameter directions were made independent of each other in order to be able to measure the minimal lumen diameter (MLD) more accurately, especially in curved vessel segments. For ostial segments, an extension of this approach was used, to allow measurement of ostial lesions in sidebranches more proximal than using conventional methods. Furthermore, two new bifurcation approaches were developed. The validation study shows that the straight segment approach results in significant smaller MLDs (on average 0.032 mm) and the ostial approach achieves on average an increase in %DS of 3.8% and an increase in lesion length of 0.59 mm due to loosening the directional constraint. The validation of our new bifurcation approaches in phantom data as well as clinical data shows only small differences between pre- and post-intervention measurements of the reference diameters outside the bifurcation core (errors smaller than 0.06 mm) and the bifurcation core area (errors smaller than 1.4% for phantom data). In summary, these new approaches have led to further improvements in the quantitative analyses of (cardio-)vascular X-ray angiographies.

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Our new bifurcation approaches including their edge segment analyses are very robust and reproducible, and therefore a great extension to the field of quantitative coronary angiography.

Over the last several years significant interest has arisen in bifurcation stenting, in particular stimulated by the European Bifurcation Club. Traditional straight vessel analysis by QCA does not satisfy the requirements for such complex morphologies anymore. To come up with practical solutions, we have developed two models, a Y-shape and a T-shape model, suitable for bifurcation QCA analysis depending on the specific anatomy of the coronary bifurcation. The principles of these models are described in this paper, as well as the results of validation studies carried out on clinical materials. It can be concluded that the accuracy, precision and applicability of these new bifurcation analyses are conform the general guidelines that have been set many years ago for conventional QCA-analyses.