Protein misfolding and formation of β-sheet-rich amyloid fibrils or aggregates is related to cellular toxicity and decay in various human disorders including Alzheimer's and Parkinson's disease. Recently, we demonstrated that the polyphenol (-)-epi-gallocatechine gallate (EGCG) inhibits α-synuclein and amyloid-β fibrillogenesis. It associates with natively unfolded polypeptides and promotes the self-assembly of unstructured oligomers of a new type. Whether EGCG disassembles preformed amyloid fibrils, however, remained unclear. Here, we show that EGCG has the ability to convert large, mature α-synuclein and amyloid-β fibrils into smaller, amorphous protein aggregates that are nontoxic to mammalian cells. Mechanistic studies revealed that the compound directly binds to β-sheetrich aggregates and mediates the conformational change without their disassembly into monomers or small diffusible oligomers. These findings suggest that EGCG is a potent remodeling agent of mature amyloid fibrils.Alzheimer | Parkinson | catechine | misfolding | oligomer P revious studies have shown that the polyphenol (-)-epi-gallocatechine gallate (EGCG), found in large amounts in green tea, has antiamyloidogenic properties and modulates the misfolding of disease proteins and prions (1-5). EGCG directly binds to unfolded polypeptide chains and inhibits β-sheet formation, an early event in the amyloid formation cascade (6). In the presence of EGCG, the assembly of a new type of unstructured, SDSstable, nontoxic oligomer was observed, instead of the expected formation of β-sheet-rich aggregates. This suggested that the compound redirects aggregation prone polypeptides into offpathway protein assemblies (6), as has since been confirmed for other flavonoids (7).These findings raise the question of whether EGCG might also be able to disassemble preformed, β-sheet-rich structures as well as earlier intermediates of fibrillogenesis. Other small molecules such as curcumin or short β-sheet breaker peptides were described to have this ability; however, their mechanism of action has not been elucidated (8,9). In the present study, we examined the ability of EGCG to alter the structure of mature amyloid fibrils with biochemical and biophysical as well as cell-based assays. Results and DiscussionTo study the effect of EGCG on preformed amyloid aggregates, we first produced α-synuclein (αS) fibrils by incubating natively unfolded monomers (100 μM) at 37°C for 7 d in phosphate buffer. Then aggregates were characterized by EM, atomic force microscopy (AFM), Thioflavin T (ThT) binding assays, and CD spectroscopy (Fig. S1). We observed that the in vitro generated αS aggregates have a β-sheet structure and a fibrillar morphology. Moreover, they efficiently bind the dye ThT, supporting previously published results (10).Next, we added an equimolar concentration of EGCG to the fibrils (50 μM αS monomer equivalent). The effect of the compound was monitored by time-resolved EM and AFM. We found that EGCG very efficiently remodels the ordered, fibrillar morphology ...

Aggrecan cleavage by a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 5 (ADAMTS-5) is crucial for the breakdown of cartilage matrix during osteoarthritis, a degenerative joint disease that leads to the progressive destruction of articular structures. The mechanisms of ADAMTS-5 activation and their links to the pathogenesis of osteoarthritis remain poorly understood, but syndecans have been shown to be involved in the activation of ADAMTS-4 (ref. 3). Here we show that syndecan-4 is specifically induced in type X collagen-producing chondrocytes both in human osteoarthritis and in murine models of the disease. The loss of syndecan-4 in genetically modified mice and intra-articular injections of syndecan-4-specific antibodies into wild-type mice protect from proteoglycan loss and thereby prevent osteoarthritic cartilage damage in a surgically induced model of osteoarthritis. The occurrence of less severe osteoarthritis-like cartilage destruction in both syndecan-4-deficient mice and syndecan-4-specific antibody-treated wild-type mice results from a marked decrease in ADAMTS-5 activity. Syndecan-4 controls the activation of ADAMTS-5 through direct interaction with the protease and through regulating mitogen-activated protein kinase (MAPK)-dependent synthesis of matrix metalloproteinase-3 (MMP-3). Our data suggest that strategies aimed at the inhibition of syndecan-4 will be of great value for the treatment of cartilage damage in osteoarthritis.

The gamma isoform of phosphoinositide 3-kinase (PI3Kgamma) has been viewed as restricted to leukocytes mediating the regulation of chemokine-induced migration and recruitment of neutrophils, monocytes, and macrophages. In line with the observation that PI3Kgamma-deficient mice display defects in adaptive immunity, inhibition of PI3Kgamma reduces synovial inflammation in the collagen-induced arthritis mouse model of inflammatory arthritis [rheumatoid arthritis (RA)], which has been attributed to reduced influx of inflammatory cells. Challenging the concept of leukocyte-restricted PI3Kgamma function, we report here a novel, nonredundant function of PI3Kgamma as an important regulator of fibroblast-induced cartilage destruction during chronic destructive arthritis. We show that in human tumor necrosis factor transgenic mice, the loss of PI3Kgamma leads to a milder inflammatory arthritis. Interestingly, PI3Kgamma deficiency does not alter the recruitment of inflammatory cells, but significantly reduces cartilage damage through reduced expression of matrix metalloproteinases in fibroblasts and chondrocytes. In vitro analyses demonstrate that the decreased invasiveness of fibroblasts is mediated by reduced phosphorylation of Akt and extracellular signal-regulated kinase. Using a PI3Kgamma specific inhibitor, these data are confirmed in human synovial fibroblasts from patients with RA who exhibit a disease-specific up-regulation of PI3Kgamma. Our data indicate that in addition to mediating the recruitment of inflammatory cells, PI3Kgamma is an important regulator of fibroblast-mediated joint destruction in RA and suggest that specific inhibitors of PI3Kgamma will interfere with the activation of RA synovial fibroblasts and reduce cartilage destruction in RA.

Introduction The rheumatoid arthritis (RA) synovium is characterised by the presence of an aggressive population of activated synovial fibroblasts (RASFs) that are prominently involved in the destruction of articular cartilage and bone. Accumulating evidence suggests that RASFs are relatively resistant to Fas-ligand (FasL)-induced apoptosis, but the data concerning tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) have been conflicting. Here, we hypothesise that the susceptibility of RASFs to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL-and TRAIL-induced programmed cell death of RASFs in vitro.

In osteoarthritis (OA), hepatocyte growth factor (HGF) is supposed to play a role in cartilage repair. Because the development of osteophytes is a major characteristic of OA and thought to be part of an attempted repair process, the purpose of this study was to determine whether HGF may be involved in osteophyte formation. HGF levels in synovial fluids from 41 patients assessed by enzyme immunosorbant assay were correlated with disease severity and osteophyte formation, evaluated by anteroposterior weight-bearing radiographs. Detection of HGF, c-Met, and CD68 in cartilage and synovial tissues was assessed by immunohistochemistry. Effects of HGF on the secretion of TGF-b1 and BMP-2 by chondrocytes, fibroblast-like synovial cells (FLS), and macrophages as well as HGF-induced secretion of MCP-1 by FLS and chondrocytes were determined by ELISA. HGF was detected in all synovial fluids and concentrations correlated highly with disease severity and osteophyte formation ( p < 0.001). Immunohistochemistry revealed weak synovial staining for HGF, whereas increasing numbers of HGF expressing chondrocytes were detected depending on disease severity. In addition, an increased number of macrophages in synovial specimens was observed, which was likewise severity dependent. In a series of subsequent in vitro studies, HGF remarkable induced MCP-1 secretion by FLS in a dose-dependent manner. No effect on TGF-b1 and BMP-2 secretion by FLS and chondrocytes was evident upon HGF stimulation, whereas secretion of these growth factors by PMA-differentiated THP-1 cells was significantly increased by HGF. The results indicate that HGF may facilitate osteophyte development by promoting MCP-1-mediated entry of monocytes/macrophages into the OA-affected joint and/or by stimulating macrophage-derived growth factors. ß