Solid phase analysis of WARP/collagen VI interaction.

<p>a, WARP binding to collagen VI tetramers. Coated collagen VI was incubated with recombinant WARP. Data are means of triplicate determinations +/− SE. <i>Inset</i>, Scatchard analysis of binding data reveals an apparent kDa of 22 nM. b, Reciprocal solid phase binding experiment showing collagen VI binding to coated WARP dimer but not WARP multimers. Representative curves are shown. c, WARP binds to both pepsinized collagen VI tetramers (black diamonds) and pepsinized intact collagen VI (white squares).</p

Collagen VI suprastructure analysis.

<p>A, Collagen VI suprastructures were isolated from human articular cartilage extracts using superparamagnetic immunobeads coupled to collagen VI antibodies. The isolated suprastructures were then doubly immuno-labeled with sheep anti-WARP antiserum (18-nm gold particles; black arrowheads) and rabbit anti-collagen VI antibody (small gold particles; white arrowheads). WARP is present in collagen VI suprastructures. Scale bar is 100 nm.</p

WARP binds to collagen VI <i>in vitro</i>.

<p>Isolated collagen VI microfibrils (shown in a) were mixed with recombinant WARP. Globular structures representing WARP are marked with arrowheads and visualized by negative staining (shown in b). Biotinylated recombinant WARP was visualized by gold labeled streptavidin as shown in panel c (5-nm gold particles). WARP is present on the collagen VI microfibrils (panel d) visible as structures close to the globular domains of the collagen VI microfibrils (bound WARP; black arrowheads). These structures are absent in the control experiments where WARP is omitted (see a). Biotinylated recombinant WARP (5-nm gold particles) bound near the junction between helical and globular domains of collagen VI microfibrils (panels e–g). Scale bars: 100 nm in a, b and d, 50 nm in e, 25 nm in c and f, and 10 nm in g.</p

Localization of WARP and collagen VI using electron microscopy.

<p>EM analysis on native supramolecular fragments isolated from human articular cartilage. Sheep antisera against WARP (18-nm gold particles; black arrow heads) (panels a–c) and a rabbit polyclonal anti-collagen VI (panels a and b) or monoclonal antibody against collagen VI (panel c) (12-nm gold particles; white arrowheads) was used. A secondary antibodies only control is shown in panel d. Scale bars: 100 nm.</p

Cryopreservation of tendon tissue using dimethyl sulfoxide combines conserved cell vitality with maintained biomechanical features

<div><p>Biomechanical research on tendon tissue evaluating new treatment strategies to frequently occurring clinical problems regarding tendon degeneration or trauma is of expanding scientific interest. In this context, storing tendon tissue deep-frozen is common practice to collect tissue and analyze it under equal conditions. The commonly used freezing medium, phosphate buffered saline, is known to damage cells and extracellular matrix in frozen state. Dimethyl sulfoxide, however, which is used for deep-frozen storage of cells in cell culture preserves cell vitality and reduces damage to the extracellular matrix during freezing. In our study, Achilles tendons of 26 male C57/Bl6 mice were randomized in five groups. Tendons were deep frozen in dimethyl sulfoxide or saline undergoing one or four freeze-thaw-cycles and compared to an unfrozen control group analyzing biomechanical properties, cell viability and collagenous structure. In electron microscopy, collagen fibrils of tendons frozen in saline appeared more irregular in shape, while dimethyl sulfoxide preserved the collagenous structure during freezing. In addition, treatment with dimethyl sulfoxide preserved cell viability visualized with an MTT-Assay, while tendons frozen in saline showed no remaining metabolic activity, indicating total destruction of cells during freezing. The biomechanical results revealed no differences between tendons frozen once in saline or dimethyl sulfoxide. However, tendons frozen four times in saline showed a significantly higher Young’s modulus over all strain rates compared to unfrozen tendons. In conclusion, dimethyl sulfoxide preserves the vitality of tendon resident cells and protects the collagenous superstructure during the freezing process resulting in maintained biomechanical properties of the tendon.</p></div

Representative ultrathin sections of articular cartilage of adult mice co-stained for WARP (18-nm gold particles; black arrow heads) and collagen VI (12-nm gold particles; white arrowheads).

<p>Scale bars: 100 nm.</p

Analysis of collagen VI, WARP and perlecan in human articular cartilage.

<p>Immuno-gold EM was conducted on native supramolecular fragments isolated from human articular cartilage for WARP (18-nm gold particles; black arrowheads), perlecan (12-nm gold particles; white arrowheads) and collagen VI (6-nm gold particles, arrows). All three components are part of complexes at the suprastructural level (shown in a). The magnified image (shown in b) shows these complexes in close contact to banded collagen fibrils. Scale bars: 100 nm (panel a) and 200 nm (panel b).</p

Immunohistochemical localization of WARP and collagen VI in articular cartilage.

<p>A, Superficial zone human articular cartilage was stained for collagen VI (panels a, d and g) and WARP (panels b, e and h). The merged images show clear co-localization of collagen VI and WARP in the pericellular environment (panels c, f and i). The scale bar shown in panel (a) is 100 µm. B, Mouse tibial articular cartilage stained with collagen VI (panels a and d) and WARP antisera (panels b and e). Merged images show co-localization of collagen VI and WARP in the chondrocyte pericellular matrix (panels c and f). Scale bar in panel (a) is 200 µm and in (d) 50 µm.</p

Transport Mechanisms and Their Pathology-Induced Regulation Govern Tyrosine Kinase Inhibitor Delivery in Rheumatoid Arthritis

<div><h3>Background</h3><p>Tyrosine kinase inhibitors (TKIs) are effective in treating malignant disorders and were lately suggested to have an impact on non-malignant diseases. However, in some inflammatory conditions like rheumatoid arthritis (RA) the <em>in vivo</em> effect seemed to be moderate. As most TKIs are taken up actively into cells by cell membrane transporters, this study aimed to evaluate the role of such transporters for the accumulation of the TKI Imatinib mesylates in RA synovial fibroblasts as well as their regulation under inflammatory conditions.</p> <h3>Methodology/Principal Findings</h3><p>The transport and accumulation of Imatinib was investigated in transporter-transfected HEK293 cells and human RA synovial fibroblasts (hRASF). Transporter expression was quantified by qRT-PCR. In transfection experiments, hMATE1 showed the highest apparent affinity for Imatinib among all known Imatinib transporters. Experiments quantifying the Imatinib uptake in the presence of specific transporter inhibitors and after siRNA knockdown of hMATE1 indeed identified hMATE1 to mediate Imatinib transport in hRASF. The anti-proliferative effect of Imatinib on PDGF stimulated hRASF was quantified by cell counting and directly correlated with the uptake activity of hMATE1. Expression of hMATE1 was investigated by Western blot and immuno-fluorescence. Imatinib transport under disease-relevant conditions, such as an altered pH and following stimulation with different cytokines, was also investigated by HPLC. The uptake was significantly reduced by an acidic extracellular pH as well as by the cytokines TNFα, IL-1β and IL-6, which all decreased the expression of hMATE1-mRNA and protein.</p> <h3>Conclusion/Significance</h3><p>The regulation of Imatinib uptake via hMATE1 in hRASF and resulting effects on their proliferation may explain moderate <em>in vivo</em> effects on RA. Moreover, our results suggest that investigating transporter mediated drug processing under normal and pathological conditions is important for developing intracellular acting drugs used in inflammatory diseases.</p> </div

hRASF actively accumulate Imatinib and express several OCTs.

<p>A) Temperature dependent uptake of Imatinib (10 µM) in hRASF at 4°C (black column) and 37°C (white column) measured by HPLC. B) Expression of investigated transporters in hRASF (white columns) and hOASF (black columns) determined by qRT-PCR. Values are mean ± SEM. n.d. = not detected.</p