The family of the CCN proteins is currently represented by 6 individual members. The eponymous members are CYR61 (Cysteine rich protein 61, CCN1), CTGF (Connective Tissue Growth Factor, CCN2) and NOV (Nephroblastoma-Overexpressed protein, CCN3). The most prominent member of this family of proteins, CTGF, has been identified as a possible biomarker for the diagnosis of fibrotic diseases. CTGF is known as a target gene and as a ‘downstream mediator’ of the TGF-beta1 signaling cascade. CTGF is involved into scarring process of organ tissues by stimulating the over expression and deposition of ECM proteins (i.e. collagens). It is reported that several cell types (e.g. endothelial cells) respond with proliferation after stimulating with CTGF. Another member of this family of proteins, NOV, has been shown as a first member of CCN protein family cell growth inhibiting properties and aberrantly reduced expression during tumourigenesis. The first reports suggest that these two CCN proteins are acting opposite to each other in regulating of ECM protein expression. Moreover, they also reciprocally influence their own expression in vitro when one of them is temporally over expressed. For the investigation of biological functions and structural details of CCN2 we have established stable transfected HEK and Flp-In™ 293 clones as productive sources for recombinant human CTGF. The overexpression of rat NOV was induced in COS-7 cells infected adenoviral vector Ad-CMV-rNOV. Both secreted and recombinant proteins were isolated from culture medium of the cells by using of heparin affinity chromatography. This method allows producing of sufficient amounts of both CCNs for structural and functional studies. The identity of purified rhCTGF and rrNOV was demonstrated by ‘Trypsin In-Gel-digesting’ followed by ESI-MS/MS mass spectrometry measurement of proteolytic peptides. The total mass of purified recombinant CCN proteins was further determined by MALDI-TOF/TOF mass spectroscopy. Biological activity of both proteins was demonstrated by using of a Smad3-sensitive reporter gene (i.e. (CAGA)12-MLP-Luc) and BrdU proliferation assay in EA hy 926 cells. Treatment with specific glycosidases further revealed that both CCN proteins are N-glycosylated confirming to bioinformatical analysis of the amino acid sequence of both CCNs. Because of the N-glycosylation both CCN-proteins differ to commercially available recombinant CNN-proteins expressed in E. coli.

The CCN family of proteins, especially its prominent member, the Connective tissue growth factor (CTGF/CCN2) has been identified as a possible biomarker for the diagnosis of fibrotic diseases. As a downstream mediator of TGF-β1 signalling, it is involved in tissue scarring, stimulates interstitial deposition of extracellular matrix proteins, and promotes proliferation of several cell types. Another member of this family, the Nephroblastoma-Overexpressed protein (NOV/CCN3), has growth-inhibiting properties. First reports further suggest that these two CCN family members act opposite to each other in regulating extracellular matrix protein expression and reciprocally influence their own expression when over-expressed. We have established stable HEK and Flp-In-293 clones as productive sources for recombinant human CCN2/CTGF. In addition, we generated an adenoviral vector for recombinant expression of rat NOV and established protocols to purify large quantities of these CCN proteins. The identity of purified human CCN2/CTGF and rat CCN3/NOV was proven by In-gel digest followed by ESI-TOF/MS mass spectrometry. The biological activity of purified proteins was demonstrated using a Smad3-sensitive reporter gene and BrdU proliferation assay in permanent cell line EA•hy 926 cells. We further demonstrate for the first time that both recombinant CCN proteins are N-glycosylated.

Liver fibrosis is the common sequel of chronic liver diseases and is associated with high morbidity and mortality in affected patients. In recent years, the contribution of chemokines and their receptors to liver fibrosis has been delineated. Chemokines are a family of chemotactic and immunomodulatory molecules that act through different G-protein coupled receptors on target cells. Apart from their classical function of regulating immune cell recruitment during chronic liver injury, chemokines can directly affect the function of hepatic stellate cells within the liver. Up to now, nine of the 19 known chemokine receptors have been characterised on stellate cells. Stimulation of most of these receptors with specific ligands leads to increased migration and proliferation of stellate cells, suggesting predominantly profibrotic effects of chemokines. The only chemokine receptor with potential antifibrotic effects identified so far is CXCR3. Notably, hepatic stellate cells are not only a target but also a source of chemokines which contributes to the direct interaction between stellate cells and other cells during fibrogenesis. The further characterisation of this interaction will yield new therapeutic options for the treatment of chronic liver diseases. In this respect chemokines are a valuable target as oral chemokine receptor antagonists have already been licensed for human use.

Despite tremendous efforts in disclosing the pathophysiological and epidemiological factors associated with liver fibrogenesis, non-invasive diagnostic measures to estimate the clinical outcome and progression of liver fibrogenesis are presently limited. Therefore, there is a mandatory need for methodologies allowing the reasonable and reliable assessment of the severity and/or progression of hepatic fibrogenesis. We here performed proteomic serum profiling by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in 179 samples of patients chronically infected with hepatitis C virus and 195 control sera. Multidimensional analysis of spectra allowed the definition of algorithms capable to distinguish class-specific protein expression profiles in serum samples. Overall about 100 peaks could be detected per single spectrum. Different algorithms including protein peaks in the range of 2000 and 10,000 Da were generated after pre-fractionation on a weak cation exchange surface. A specificity of 93% with a sensitivity of 86% as mean of the test set results was found, respectively. The nature of three of these protein peaks that belonged to kininogen-1 and thymosin-β(4) was further analysed by tandem mass spectrometry (MS)/MS. We further found that kininogen-1 mRNA was significantly down-regulated in cirrhotic livers. We have identified kininogen-1 and thymosin-β(4) as potential new biomarkers for human chronic hepatitis C and conclude that serum profiling is a reliable technique to identify hepatitis-associated expression patterns. Based on the high throughput capability, the identified differential protein panel may serve as a diagnostic marker and warrants further validation in larger cohorts.

Background: CRP2 and FHL2 belong to the superfamily of LIM-domain proteins that are characterized by a special arrangement of zinc-fingers that are necessary for protein-protein interactions [1]. CRP2 has two and FHL2 has four and a half LIM domains. They are implicated in wound healing and assembly of extracellular matrix proteins [2]. In the liver, the expression of CRP2 and FHL2 is restricted to HSC. The main aim of the present investigation is to identify the signalling pathways that drive the expression of the respective genes in the process of transdifferentation and to analyse if this expression or the cellular localisation of these LIM domain proteins is modulated by TGF-β or Sphingosine-1-phosphate (S1P).

CCN proteins are small secreted cysteine-rich proteins containing up to four individual structural modules including an insulin-like growth factor binding domain, a von Willebrand Factor type C motif, a thrombospondin type I module and a carboxyl-terminal cystine knot. Actually, there is a large body of evidence suggesting that members of the CCN protein family encompass an expansive repertoire of functions in crucial areas including control of development, cell fate, angiogenesis, tumorigenesis, osteogenesis, cell adhesion, mitogenesis, migration, chemotaxis, and cell survival. Moreover, this family is supposed to modulate signalling of integrins, transforming growth factor-betas, bone morphogenetic proteins, vascular endothelial growth factor, Notch and factors that mediate signals via the canonical Wingless-type MMTV integration site family. However, several of these properties are not substantiated by experimental data but were deduced from proteins sharing one or more of the structural modules with these proteins. In this review, the actual knowledge of biological activities and molecular involvement of CCN proteins in maintenance of liver health and in initiation and progression of hepatic diseases is summarized and discussed.

E-Type cyclins E1 (CcnE1) and E2 (CcnE2) are important mediators for driving resting cells into the proliferative state of the cell cycle but are dispensable in continuously dividing cells. We recently showed that CcnE1 is a key player for hepatocyte proliferation during liver regeneration, whereas CcnE2 is essentially a regulator of CcnE1. Liver fibrogenesis is associated with proliferation of hepatocytes and hepatic stellate cells (HSC). Thus, the aim of this study was to investigate the potential contribution of CcnE1 for liver fibrosis in human and mice.

Abstract Introduction Hyperglycemia and insulin resistance are well-known features of critical illness and impact the mortality rate, especially in sepsis. Retinol binding protein 4 (RBP4) promotes insulin resistance in mice and is systemically elevated in patients with obesity and type 2 diabetes. We investigated the potential role of RBP4 in critically ill patients. Methods We conducted a prospective single-center study of serum RBP4 concentrations in critically ill patients. One hundred twenty-three patients (85 with sepsis, 38 without sepsis) were studied at admission to a medical intensive care unit (ICU) before initiation of specific intensive care treatment measures and compared to 42 healthy nondiabetic controls. Clinical data, various laboratory parameters and metabolic and endocrine functions were assessed. Patients were followed for approximately 3 years. Results Serum RBP4 was significantly reduced in ICU patients, independently of sepsis, as compared to healthy controls ( P < 0.001). Patients with liver cirrhosis as the primary underlying diagnosis for ICU admission had significantly lower RBP4 levels as compared with other ICU patients. Accordingly, in all ICU patients, serum RBP4 closely correlated with liver function and increased with renal failure. No significant differences of serum RBP4 concentrations in septic patients with pulmonary or other origins of sepsis or nonseptic patients could be revealed. Acute phase proteins were inversely correlated with RBP4 in sepsis patients. RBP4 did not differ between patients with or without obesity or preexisting diabetes. However, serum RBP4 levels correlated with endogenous insulin secretion (C-peptide) and insulin resistance (HOMA index). Low serum RBP4 upon admission was an adverse predictor of short-term survival in the ICU, but was not associated with overall survival during long-term follow-up. Conclusions Serum RBP4 concentrations are significantly reduced in critically ill patients. The strong associations with hepatic and renal function, insulin resistance and acute mortality collectively suggest a role of RBP4 in the pathogenesis of critical illness, possibly as a negative acute phase reactant, and allow a proposition as a potential novel biomarker for ICU patients.