Christian Freise

The alpha 2 chain of collagen type VI sequesters latent proforms of matrix-metalloproteinases and modulates their activation and activity.

The extracellular matrix (ECM) attracts increasing attention as a store of biologically active molecules and as a reservoir of potent cell signalling molecules released by proteolytic action. Both, cytokines and proteases mediating such release are sequestered in the ECM. Here, we found matrix metalloproteinase (MMP) proforms closely associated with collagenous septae in fibrotic liver tissue, and we screened immobilized human placenta-derived collagen chains and other ECM proteins for MMP-binding activity. Following the establishment of a novel highly-efficient two-step chromatography procedure for the isolation of the purified alpha-chains of the pepsin-resistant triple-helical CVI fragment (CVI/PR) solid phase and surface plasmon resonance binding studies were performed. We identified the triple-helical domain of the alpha2 chain of microfilamentous CVI alpha2(VI) as having nanomolar affinity for the collagenases proMMP-1, -8 , -13 and stromelysin-1 (MMP-3), thus extending the repertoire of pericellular and substrate-based interactions of MMPs. Enzymatic activity assays enabled the correlation of MMP activity with CVI binding, in that alpha2(VI) chain-mediated inhibition of enzymatic activity is accompanied by increased binding. Similar results were shown for the gelatinase proMMP-9, whereas for proMMP-2, the alpha2(VI) chain at low concentrations seems to interfere with prodomain binding resulting in enhanced activity without scission of the prodomain. Stable complexes of proMMP-2 and alpha2(VI) chain competed with gelatinase binding to the preferred ligand, collagen type I. In conclusion, the alpha2(VI) chain modulates MMP availability by sequestering proMMPs in the ECM, blocking proteolytic activity. Therefore, CVI and especially its alpha2(VI) chain might serve as a lead structure for MMP-based therapeutics which modulates the action of these matrix components, e.g. in fibrosis and cancer.

Hydroxyproline-containing collagen analogs trigger the release and activation of collagen-sequestered proMMP-2 by competition with prodomain-derived peptide P33-42

BackgroundFibrolytic and profibrotic activities of the matrix metalloproteinases (MMPs)-2 and -9 play a central role in liver fibrosis. Since binding to the extracellular matrix influences the activity of both gelatinases, here the role of fibrillar collagens as the most abundant matrix components in fibrotic tissue was investigated.ResultsIn situ zymography and immunohistology showed association of enzymatically inactive prodomain-containing proMMP-2 and proMMP-9 but not of their activated forms to fibrillar collagen structures, which are not substrates of these gelatinases. In solid-phase binding studies with human collagens and collagen fragments, up to 45% of [125I]-labeled proMMP-2 and proMMP-9 but not of active (act)MMP-2 and actMMP-9 were retained by natural collagenous molecules and by synthetic analogs containing repeated Gly-Pro-Hyp triplets (GPO). Surface plasmon resonance yielded binding constants for the interaction of collagen type I (CI) with proMMP-2 and proMMP-9 in a nanomolar range. Values for actMMP-2 and actMMP-9 were 30-40 times higher. Tenfold molar excesses of (GPO)10 reduced the interaction of CI with pro- and actMMP-2 by 22- or 380-fold and resulted in prodomain release accompanied by high enzymatic activation and activity. Pointing to gelatine substrate displacement, higher (GPO)10 concentrations blocked the enzymatic activity. The MMP-2 prodomain-derived collagen-binding domain peptide (P33-42) binds to the collagen-binding domain of MMP-2, thereby preserving enzymatic inactivity. Synthetic P33-42 peptide competed with proMMP-2 binding to CI and prevented (GPO)10-mediated proMMP-2 activation. In contrast to (GPO)10, P33-42 did not activate proMMP-2, making triple helical and hydroxyproline-containing (GPO)10 unique in modulating gelatinase availability and activity.ConclusionsThese findings suggest novel strategies using collagen analogs for the resolution of liver fibrosis via fibrotic matrix-sequestered gelatinases.

An active extract of Lindera obtusiloba inhibits adipogenesis via sustained Wnt signaling and exerts anti-inflammatory effects in the 3T3-L1 preadipocytes

Obesity, the related metabolic syndrome and associated liver diseases represent an epidemic problem and demand for effective therapeutic strategies. In this regard, natural compounds derived from Oriental medicine such as green tea polyphenols influencing adipogenesis attract growing attention. In Korea, an aqueous extract from the Japanese spice bush Lindera obtusiloba is traditionally used for treatment of inflammation and prevention of liver damage. We here investigated effects of the L. obtusiloba extract on cell growth, apoptosis, Wnt signaling and differentiation of (im)mature adipocytes using 3T3-L1, an established cell line for studying adipogenesis. L. obtusiloba extract reduced the de novo DNA synthesis of 3T3-L1 preadipocytes in a concentration dependent manner with an IC(50) of ∼135 μg/ml paralleled by induction of caspase 3/7 mediated apoptosis. Hormone-induced 3T3 L1 differentiation in the presence of L. obtusiloba extract resulted in a reduced accumulation of intracellular lipid droplets by 70%, in down-regulated expression of the adipogenesis-associated proteins glucose transporter-4 and vascular endothelial growth factor, in reduced secretion of the proadipogenic matrix metalloproteinase-2, and in dampened phosphorylation of the Wnt pathway effector protein β-catenin with subsequent diminished expression of the peroxisome proliferator-activated receptor-γ. Treatment of mature adipocytes with L. obtusiloba extract also significantly reduced intracellular lipid droplets. In addition to this strong interference of L. obtusiloba extract with adipogenesis, L. obtusiloba extract exerted anti-inflammatory effects. L. obtusiloba extract significantly attenuated lipopolysaccharide- and tumor necrosis factor α-induced secretion of IL-6 by preadipocytes, thus influencing insulin resistance and inflammatory state characterizing obesity. In conclusion, extracts of L. obtusiloba should be evaluated as a potential complementary treatment option for obesity associated with the metabolic syndrome.

The Elongated First Fibronectin Type III Domain of Collagen XIV Is an Inducer of Quiescence and Differentiation in Fibroblasts and Preadipocytes

Collagen XIV (CXIV) is a fibril-associated collagen that is mainly expressed in well differentiated tissues and in late embryonic development. Because CXIV is almost absent in proliferating and/or dedifferentiated tissues, a functional role in maintaining cell differentiation is suspected. We demonstrate antiproliferative, quiescence- and differentiation-inducing effects of human CXIV and its recombinant fragments on mesenchymal cells. In primary human fibroblasts, in mouse 3T3 fibroblasts and in 3T3-L1 preadipocytes, CXIV reduced de novo DNA synthesis by 75%, whereas cell numbers and viability remained unaltered. Cells showed no signs of apoptosis, and maximal proliferation was restored when serum was supplemented, thus indicating that CXIV induced reversible cellular quiescence. Exposure of fibroblasts to CXIV in vitro led to cellular bundles and clusters. CXIV also triggered trans-differentiation of 3T3-L1 preadipocytes into adipocytes, as could be shown by lipid accumulation and by expression of the glucose transporter Glut4. These effects were also observed with the amino-terminal recombinant fragment Gln29-Pro154 that harbors the first fibronectin type III domain and a 39-amino-acid extension, whereas no activity was found for all other recombinant CXIV fragments. Based on these finding the development of small molecular analogs that modulate fibroblast cell growth and differentiation, e.g. in wound healing and fibrosis, seems feasible.

A hepatoprotective Lindera obtusiloba extract suppresses growth and attenuates insulin like growth factor-1 receptor signaling and NF-kappaB activity in human liver cancer cell lines

BackgroundIn traditional Chinese and Korean medicine, an aqueous extract derived from wood and bark of the Japanese spice bush Lindera obtusiloba (L.obtusiloba) is applied to treat inflammations and chronic liver diseases including hepatocellular carcinoma. We previously demonstrated anti-fibrotic effects of L.obtusiloba extract in hepatic stellate cells. Thus, we here consequently examine anti-neoplastic effects of L.obtusiloba extract on human hepatocellular carcinoma (HCC) cell lines and the signaling pathways involved.MethodsFour human HCC cell lines representing diverse stages of differentiation were treated with L.obtusiloba extract, standardized according to its known suppressive effects on proliferation and TGF-β-expression. Beside measurement of proliferation, invasion and apoptosis, effects on signal transduction and NF-κB-activity were determined.ResultsL.obtusiloba extract inhibited proliferation and induced apoptosis in all HCC cell lines and provoked a reduced basal and IGF-1-induced activation of the IGF-1R signaling cascade and a reduced transcriptional NF-κB-activity, particularly in the poorly differentiated SK-Hep1 cells. Pointing to anti-angiogenic effects, L.obtusiloba extract attenuated the basal and IGF-1-induced expression of hypoxia inducible factor-1α, vascular endothelial growth factor, peroxisome proliferator-activated receptor-γ, cyclooxygenase-2 and inducible nitric oxide synthase.ConclusionsThe traditional application of the extract is confirmed by our experimental data. Due to its potential to inhibit critical receptor tyrosine kinases involved in HCC progression via the IGF-1 signaling pathway and NF-κB, the standardized L.obtusiloba extract should be further analysed for its active compounds and explored as (complementary) treatment option for HCC.

Extracts of Lindera obtusiloba induce antifibrotic effects in hepatic stellate cells via suppression of a TGF-β-mediated profibrotic gene expression pattern ☆

Liver fibrosis is characterized by high expression of the key profibrogenic cytokine transforming growth factor (TGF)-beta and the natural tissue inhibitor of metalloproteinases (TIMP)-1, leading to substantial accumulation of extracellular matrix. Liver fibrosis originates from various chronic liver diseases, such as chronic viral hepatitis that, to date, cannot be treated sufficiently. Thus, novel therapeutics, for example, those derived from Oriental medicine, have gained growing attention. In Korea, extracts prepared from Lindera obtusiloba are used for centuries for treatment of inflammation, improvement of blood circulation and prevention of liver damage, but experimental evidence of their efficacy is lacking. We studied direct antifibrotic effects in activated hepatic stellate cells (HSCs), the main target cell in the fibrotic liver. L. obtusiloba extract (135 mug/ml) reduced the de novo DNA synthesis of activated rat and human HSCs by about 90%, which was not accompanied by cytotoxicity of HSC, primary hepatocytes and HepG2 cells, pointing to induction of cellular quiescence. As determined by quantitative polymerase chain reaction, simultaneous treatment of HSCs with TGF-beta and L. obtusiloba extract resulted in reduction of TIMP-1 expression to baseline level, disruption of the autocrine loop of TGF-beta autoinduction and increased expression of fibrolytic matrix metalloproteinase (MMP)-3. In addition, L. obtusiloba reduced gelatinolytic activity of HSC by interfering with profibrogenic MMP-2 activity. Since L. obtusiloba extract prevented intracellular oxidative stress experimentally induced by tert-butylhydroperoxide, we concluded that the direct antifibrotic effect of L. obtusiloba extract might be mediated by antioxidant activity. Thus, L. obtusiloba, traditionally used in Oriental medicine, may complement treatment of chronic liver disease.

Inhibition of vascular calcification by block of intermediate conductance calcium-activated potassium channels with TRAM-34.

Vascular calcifications are a hallmark of advanced cardiovascular disease in patients with chronic kidney disease. A key event is the transition of contractile vascular smooth muscle cells (VSMC) into an osteoblast-like phenotype, promoting a coordinated process of vascular remodeling resembling bone mineralization. Intermediate-conductance calcium-activated potassium channels (KCa3.1) are expressed in various tissues including VSMC. Aiming for novel therapeutic targets in vascular calcification, we here studied effects of KCa3.1-inhibition on VSMC calcification by the specific KCa3.1 inhibitor TRAM-34. Calcification in the murine VSMC cell line MOVAS-1 and primary rat VSMC was induced by calcification medium (CM) containing elevated levels of PO4(3-) and Ca(2+). Cell signaling, calcification markers, and release of nitric oxide and alkaline phosphatase were assessed by luciferase reporter plasmids, RT-PCR and specific enzymatic assays, respectively. KCa3.1 gene silencing was achieved by siRNA experiments. TRAM-34 at 10nmol/l, decreased CM-induced calcification and induced NO release of VSMC accompanied by decreased TGF-β signaling. The CM-induced mRNA expressions of osterix, osteocalcin, matrix-metalloproteinases (MMP)-2/-9 were reduced by TRAM-34 while osteopontin expression was increased. Further, TRAM-34 attenuated the CM- and TNF-α-induced activation of NF-κB and reduced the release of MMP-2/-9 by VSMC. Finally, TRAM-34 abrogated CM-induced apoptosis and KCa3.1 gene silencing protected VSMC from CM-induced onset of calcification. In summary, TRAM-34 interferes with calcification relevant signaling of NF-κB and TGF-β thereby blocking the phenotypic transition/calcification of VSMC. We conclude that the results provide a rationale for further studies regarding a possible therapeutic role of KCa3.1 inhibition by TRAM-34 or other inhibitors in vascular calcification.

(+)-Episesamin inhibits adipogenesis and exerts anti-inflammatory effects in 3T3-L1 (pre)adipocytes by sustained Wnt signaling, down-regulation of PPARγ and induction of iNOS.

Obesity and its associated health risks still demand for effective therapeutic strategies. Drugs and compositions derived from Oriental medicine such as green tea polyphenols attract growing attention. Previously, an extract from the Japanese spice bush Lindera obtusiloba (L. obtusiloba) traditionally used for treatment of inflammation and prevention of liver damage was shown to inhibit adipogenesis. Aiming for the active principle of this extract (+)-episesamin was identified, isolated and applied in adipogenic research using 3T3-L1 (pre)adipocytes, an established cell line for studying adipogenesis. With an IC50 of 10μM (+)-episesamin effectively reduced the growth of 3T3-L1 preadipocytes and decreased hormone-induced 3T3-L1 differentiation as shown by reduced accumulation of intracellular lipid droplets and diminished protein expression of GLUT-4 and vascular endothelial growth factor. Mechanistically, the presence of (+)-episesamin during hormone-induced differentiation provoked a reduced phosphorylation of ERK1/2 and β-catenin along with a reduced protein expression of peroxisome proliferator-activated receptor γ and a strongly increased protein expression of iNOS. Treatment of mature adipocytes with (+)-episesamin resulted in a reduction of intracellular stored lipid droplets and induced the proapoptotic enzymes caspases-3/-7. Besides interfering with adipogenesis, (+)-episesamin showed anti-inflammatory activity by counteracting the lipopolysaccharide- and tumor necrosis factor α-induced secretion of interleukin 6 by 3T3-L1 preadipocytes. In conclusion, (+)-episesamin seems to be the active drug in the L. obtusiloba extract being responsible for the inhibition of adipogenesis and, thus, should be evaluated as a novel potential complementary treatment for obesity.

K+-channel inhibition reduces portal perfusion pressure in fibrotic rats and fibrosis associated characteristics of hepatic stellate cells

In liver fibrosis, activated hepatic stellate cells (HSC) secrete excess extracellular matrix, thus, represent key targets for antifibrotic treatment strategies. Intermediate‐conductance Ca2 + ‐activated K+‐channels (KCa3.1) are expressed in non‐excitable tissues affecting proliferation, migration and vascular resistance rendering KCa3.1 potential targets in liver fibrosis. So far, no information about KCa3.1 expression and their role in HSC exists. Aim was to quantify the KCa3.1 expression in HSC depending on HSC activation and investigation of antifibrotic properties of the specific KCa3.1 inhibitor TRAM‐34 in vitro and in vivo.

The synthetic hydroxyproline-containing collagen analogue (Gly-Pro-Hyp)10 promotes enzymatic activity of matrixmetalloproteinase-2 in vitro

Diseases such as liver fibrosis and intestinal inflammation are characterized by accumulated components of the extracellular matrix (ECM). Given that fibrillar collagen structures were shown to serve as storage site for inactive proforms of matrixmetalloproteinases (MMPs), modulating this MMP-collagen interaction might offer a rational interventional (therapeutic) approach to enhance degradation of accumulated ECM. The synthetic triple helical collagen analogue (Gly-Pro-Hyp)10 - (GPO)10 - was shown to trigger release and enzymatic activation of collagen sequestered proMMP-2. In the presented study, we, for the first time, investigated how MMP-(GPO)10 interaction impacts cellular responses in vitro. We found that recombinant proMMP-2 induced proliferation of hepatic stellate cells (HSC), which was enhanced after addition of (GPO)10 reaching comparable levels following incubation with fully activated MMP-2. In addition, (GPO)10 induced HSC migration similar to the platelet-derived growth factor subunit-B. Further, the MMP-2-dependent invasion of HT1080 fibrosarcoma cells through an ECM membrane was enhanced after addition of (GPO)10. Since cellular proliferation and migration concomitant with matrix degradation is stimulated, we conclude that the MMP-(GPO)10 interaction also functions in a physiological environment. Thus, a potential therapeutic effect of (GPO)10 should be further tested in animal models for MMP-associated diseases such as colitis or fibrosis.