Hydrolyzed fish collagen induced chondrogenic differentiation of equine adipose tissue-derived stromal cells.

Adipose-derived stromal cells (ADSCs) are multipotent cells which, in the presence of appropriate stimuli, can differentiate into various lineages such as the osteogenic, adipogenic and chondrogenic. In this study, we investigated the effect of transforming growth factor beta 1 (TGF-ß1) in comparison to hydrolyzed fish collagen in terms of the chondrogenic differentiation potential of ADSCs. ADSCs were isolated from subcutaneous fat of horses by liposuction. Chondrogenesis was investigated using a pellet culture system. The differentiation medium was either supplemented with TGF-ß1 (5 ng/ml) or fish collagen (0.5 mg/ml) for a 3 week period. After the 3 weeks in vitro differentiation, RT-PCR and histological staining for proteoglycan synthesis and type II collagen were performed to evaluate the degree of chondrogenic differentiation and the formation of cartilaginous extracellular matrix (ECM). The differentiation of ADSCs induced by TGF-ß1 showed a high expression of glycosaminoglycan (GAG). Histological analysis of cultures stimulated by hydrolyzed fish collagen demonstrated an even higher GAG expression than cultures stimulated under standard conditions by TGF-ß1. The expression of cartilage-specific type II collagen and Sox9 was about the same in both stimulated cultures. In this study, chondrogenesis was as effectively induced by hydrolyzed fish collagen as it was successfully induced by TGF-ß1. These findings demonstrated that hydrolyzed fish collagen alone has the potential to induce and maintain ADSCs-derived chondrogenesis. These results support the application of ADSCs in equine veterinary tissue engineering, especially for cartilage repair.

[Matrix metalloproteinases in inflammatory bowel disease - from basic research to clinical significance]. / Matrix-Metalloproteinasen bei chronisch-entzündlichen Darmerkrankungen - von der Grundlagenforschung zur klinischen Bedeutung.

Matrix Metalloproteinases (MMPs) are a family of Zn (2 +)-dependent endopeptidases that are considered to be the most potent proteases in the turnover of the extracellular matrix (ECM). In addition to their capability for degradating virtually all protein components of the ECM, MMPs regulate a variety of non-matrix substrates such as chemokines, cytokines and growth factors. Therefore MMPs play a central role in a variety of physiological and pathological processes such as angiogenesis, wound healing and inflammatory response including mucosal inflammation associated with inflammatory bowel disease (IBD). Apart from mucosal destruction in IBD, recent studies have identified several new functions of MMPs for the pathophysiology of the healthy and inflamed intestine. This article summarises the main activities of MMPs in IBD with emphasis on their pathophysiological relevance and potential clinical implications based on the expression and regulation patterns of these enzymes.

MMP-9-hemopexin domain hampers adhesion and migration of colorectal cancer cells.

Matrix metalloproteinases (MMPs), in particular MMP-2 and MMP-9, are involved in colon cancer progression and metastasis due to their ability to degrade extracellular matrix (ECM) components. In previous studies we described the MMP-9 hemopexin like domain (MMP-9-PEX) as an MMP-9 antagonist. In the present study it was examined whether recombinant MMP-9-PEX has an inhibitory effect on migration and adhesion of colorectal carcinoma cells. Furthermore, we searched for MMP-9 substrate binding sites within the MMP-9-PEX by surface plasmon resonance. Migration of SW620 and LS174 cells was investigated in a modified Boyden chamber assay. In the presence of 0.2 microg/ml MMP-9-PEX migration of SW620 was decreased by 34%, while addition of 0.4 microg/ml diminished migration by 56%. Migration of LS174 cells was not affected by MMP-9-PEX. Adhesion studies were performed on 96-well plates coated with gelatin, collagen type I, and laminin, respectively. In the presence of MMP-9-PEX, adhesion of SW620 cells to these coating substrates was significantly inhibited. Surface plasmon resonance studies revealed binding of collagen type I and IV, elastin, and fibrinogen to proMMP-9 as well as to MMP-9-PEX. However, equilibrium constants (Kd) indicated a higher affinity of proMMP-9 to the matrix proteins. This could indicate that there is more than one binding site for matrix components within the entire proMMP-9 molecule. Since migration and adhesion of metastatic colorectal carcinoma cells were reduced by MMP-9-PEX, this recombinant MMP-9 antagonist might be of therapeutical interest.

Mechanisms of fibrinolysis in chronic liver injury (with special emphasis on MMPs and TIMPs).

Regeneration from liver fibrosis is characterized by four essential events: 1. eradication of pathological agents, 2. apoptosis of activated hepatic stellate cells, 3. remodeling of extracellular matrix, and 4. regeneration of parenchyma and liver function. The temporal and spatial regulation of matrix metalloproteinase (MMP) activity and expression of their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), play a pivotal role in matrix remodeling during hepatic fibrogenesis and recovery. According to current knowledge, the main topics and mechanisms in regeneration from hepatic fibrosis with special emphasis on MMPs and TIMPs are presented. MMP and TIMP expression patterns during hepatic fibrogenesis and fibrolysis, specific characteristics like regulation, expression of cell types, gene expression (RNA/protein), and the underlying disease are summarized. Studies presenting a time course for MMP and TIMP expression during recovery from hepatic fibrosis were taken into consideration to point out a synchronizing behavior in the expression pattern.

Identification of fibrosis-relevant proteins using DIGE (difference in gel electrophoresis) in different models of hepatic fibrosis.

Proteomics became a more and more important technique for the large-scale analysis of proteins during the last years. Two-dimensional (2D) electrophoresis as a major tool of proteomics is a powerful method to compare two different biological stages (e. g. healthy and diseased tissue) and to find differences in their protein pattern. One major problem in proteomics is the gel to gel variation of two-dimensional gel electrophoresis, which could cause artefacts in the detection of expression differences. The "difference in gel electrophoresis" (DIGE) technique allows the separation of two proteomes in the same gel. The protein pools were labelled with different fluorescent dyes and equal amounts of protein were separated in the same gel. Another advantage of DIGE is the possibility to separate an internal standard labelled with a third dye in the same gel to allow quantitative expression analysis. We compared proteomes of three different fibrosis models with the appropriate control (tissue inhibitor of metalloproteinases-1 (TIMP-1) overexpressing HepG2 cells in comparison to a HepG2 control, freshly isolated HSC in comparison to activated HSC and healthy mouse liver in comparison to fibrotic mouse liver). Among the differentially expressed proteins several were already found to be relevant for fibrosis but we also detected some proteins like the selenium binding protein 2 which might be relevant for hepatic fibrosis.

[Confocal laser scanning microscopy: a deep look into the cell]. / Konfokale Laserscanning-Mikroskopie: Der Blick in die Zelle.

Confocal laser scanning microscopy (CLSM) is a powerful technology for assaying biomolecular distribution and dynamics in cells and tissues. Innovations in CLSM-techniques, coupled with the development of new dyes and genetically encoded indicators, have increased both in vitro and in vivo imaging approaches. CLSM has had wide application in basic science, but little impact so far on medical investigations. As a "cutting edge" technology CLSM has proved to be a valuable tool in some areas within medical applications including pathology, dermatology, ophthalmology and research in various other fields of medicine. This paper gives an overview about the wide range of CLSM-applications and shows the enormous potential of this technology in medical research and development.

Expression of human membrane type 1 matrix metalloproteinase in Pichia pastoris.

A soluble, C-terminal truncated form of human membrane type 1 matrix metalloproteinase (MT1-MMP) containing the hemopexin-like domain was expressed in Pichia pastoris strain KM71. High levels of secreted protein were detected. Although the c-DNA for the proenzyme (Ala(21)-Glu(523) called DeltaTM-MT1-MMP) was cloned, almost only active MT1-MMP (Tyr(112)-Glu(523)) with identical N-terminus as described for the wild-type enzyme was isolated. This active enzyme was highly purified and characterized with respect to its biochemical properties. The recombinant protein showed high stability against autolysis and proteolysis by yeast proteases, although the calculated in vivo half-life is rather low. The biochemical properties of this new MT1-MMP species were compared with the well-characterized catalytic domain (Ile(114)-Ile(318)) of MT1-MMP. The novel form of MT1-MMP exhibited a higher stability against autolysis than the isolated catalytic domain (Ile(114)-Ile(318)).