Showing papers by "Martin Lotz published in 2008"

Human Endothelial Cells Produce IL-6

Abstract: The interaction between human endothelial cells and leukocytes during immunological and inflammatory responses is in part mediated through the release of soluble mediators. We report that cultured human umbilical vein endothelial cells secrete IL-6 when stimulated with lipopolysaccharide. The monokines, IL-1 and TNF-alpha, were potent inducers of IL-6, whereas lymphotoxin was only effective at much higher concentrations. IFN gamma also was a strong stimulus of IL-6 production, but TGF-beta did not have an effect at doses modulating other endothelial cell functions. Endothelial cell IL-6 was active as hybridoma-plasmacytoma growth factor and as B-cell and hepatocyte stimulating factor. Endothelial IL-6 activity was neutralized by a specific antibody to IL-6 and it was shown by immunoprecipitation to be identical in size to human fibroblast-derived IL-6. IL-6 did not have a detectable effect on several endothelial cell functions, including proliferation, adherence of leukocytes, and synthesis of PGE2, TPA, and PAI-1. As IL-6 is probably an important regulator of host defense responses, production of this cytokine by endothelial cells may contribute to the pathogenesis of various inflammatory and immunologic diseases.

Biomechanics of cartilage articulation: effects of lubrication and degeneration on shear deformation.

Abstract: Objective To characterize cartilage shear strain during articulation, and the effects of lubrication and degeneration. Methods Human osteochondral cores from lateral femoral condyles, characterized as normal or mildly degenerated based on surface structure, were selected. Under video microscopy, pairs of osteochondral blocks from each core were apposed, compressed 15%, and subjected to relative lateral motion with synovial fluid (SF) or phosphate buffered saline (PBS) as lubricant. When cartilage surfaces began to slide steadily, shear strain (Exz) and modulus (G) overall in the full tissue thickness and also as a function of depth from the surface were determined. Results In normal tissue with SF as lubricant, Exz was highest (0.056) near the articular surface and diminished monotonically with depth, with an overall average Exz of 0.028. In degenerated cartilage with SF as lubricant, Exz near the surface (0.28) was 5-fold that of normal cartilage and localized there, with an overall Exz of 0.041. With PBS as lubricant, Exz values near the articular surface were ∼50% higher than those observed with SF, and overall Exz was 0.045 and 0.062 in normal and degenerated tissue, respectively. Near the articular surface, G was lower with degeneration (0.06 MPa, versus 0.18 MPa in normal cartilage). In both normal and degenerated cartilage, G increased with tissue depth to 3–4 MPa, with an overall G of 0.26–0.32 MPa. Conclusion During articulation, peak cartilage shear is highest near the articular surface and decreases markedly with depth. With degeneration and diminished lubrication, the markedly increased cartilage shear near the articular surface may contribute to progressive cartilage deterioration and osteoarthritis.

Repression of chondrogenesis through binding of notch signaling proteins HES-1 and HEY-1 to N-box domains in the COL2A1 enhancer site.

Abstract: Objective—Notch signaling is implicated in repressing mesenchymal stem cell (MSC) chondrogenic differentiation. The mechanism of this repression and how this is modulated to permit chondrogenesis has not been elucidated. Methods—Notch intracellular domain (NICD) protein levels were monitored via western blot throughout chondrogenic differentiation of human in MSC pellet cultures. Overexpression of Notch signaling components and their effect on chondrogenesis was achieved by transfecting plasmids coding for NICD, hairy and enhancer of split 1 (Hes-1) and hairy and enhancer of split related-2 (HERP-2/Hey-1). Col2a1 and aggrecan expression was monitored via quantitative PCR. Chromatin immunoprecipitation (chIP) was utilized to test whether Hes-1 and Hey-1 bind putative N-box domains in intron 1 of Col2a1. Results—NICD protein levels were reduced during chondrogenesis of hMSC, which was mediated by TGFβ3. Col2a1 gene expression was repressed following overexpression of NICD (2-fold), Hes-1 (3-fold) and markedly by Hey-1 (80-fold). Hey-1 repressed aggrecan expression 10-fold, while NICD and Hes-1 had no effect. chIP studies show that endogenous Hes-1 and Hey-1 bind to two putative N-box domains adjacent to and as part of the Sox9 enhancer binding site located in intron 1 of Col2a1. The Hes-1 co-repressor protein transducin like enhancer (TLE) was displaced during chondrogenic differentiation and following TGFβ3 treatment. Conclusion—These results reveal novel mechanisms by which Notch signaling represses gene expression and further define the role of TGFβ to promote chondrogenic differentiation.

Expression of novel extracellular sulfatases Sulf-1 and Sulf-2 in normal and osteoarthritic articular cartilage

Abstract: Changes in sulfation of cartilage glycosaminoglycans as mediated by sulfatases can regulate growth factor signaling. The aim of this study was to analyze expression patterns of recently identified extracellular sulfatases Sulf-1 and Sulf-2 in articular cartilage and chondrocytes. Sulf-1 and Sulf-2 expressions in human articular cartilage from normal donors and patients with osteoarthritis (OA) and in normal and aged mouse joints were analyzed by real-time polymerase chain reaction, immunohistochemistry, and Western blotting. In normal articular cartilage, Sulf-1 and Sulf-2 mRNAs and proteins were expressed predominantly in the superficial zone. OA cartilage showed significantly higher Sulf-1 and Sulf-2 mRNA expression as compared with normal human articular cartilage. Sulf protein expression in OA cartilage was prominent in the cell clusters. Western blotting revealed a profound increase in Sulf protein levels in human OA cartilage. In normal mouse joints, Sulf expression was similar to human cartilage, and with increasing age, there was a marked upregulation of Sulf. The results show low levels of Sulf expression, restricted to the superficial zone in normal articular cartilage. Sulf mRNA and protein levels are increased in aging and OA cartilage. This increased Sulf expression may change the sulfation patterns of heparan sulfate proteoglycans and growth factor activities and thus contribute to abnormal chondrocyte activation and cartilage degradation in OA.

The effect of glycosaminoglycan loss on chondrocyte viability: A study on porcine cartilage explants

Abstract: Objective Loss of glycosaminoglycan (GAG) is an early event in osteoarthritis. Recent findings showed increased cell death in arthritic cartilage and linkage with extracellular matrix degradation. The aim of this study was to analyze the direct effect of GAG loss on chondrocyte survival and cell death following mechanical injury. Methods In full-thickness cartilage explants from porcine knee joints, GAG was depleted by digestion with chondroitinase ABC. Explants were subjected to single-impact mechanical injury. Cell viability and the types of cell death were analyzed by Live/Dead cell assay, staining for active caspase 3, and sensitivity to caspase inhibitor. Results GAG depletion did not directly lead to increased cell death. In chondroitinase ABC–treated explants, but not in control explants, mechanical injury caused an immediate reduction in cell viability (from 84.6% to 71.0%); the reduction was prominent in the superficial zone. This immediate cell death was not inhibited by the pancaspase inhibitor Z-VAD-FMK, suggesting cell necrosis. During subsequent culture, viability in these explants decreased further, to 50.5% on day 3. The second wave of cell death was reduced by the addition of Z-VAD-FMK in chondroitinase ABC–treated explants and was also associated with activation of caspase 3, suggesting apoptotic mechanisms of cell death. Conclusion These results indicate that GAG loss alone does not directly lead to chondrocyte death. In response to mechanical injury, there is an immediate induction of necrotic cell death that is seen only in GAG-depleted explants and primarily in the superficial zone. During subsequent culture, cell death spreads via apoptotic mechanisms.

Rho kinase–dependent CCL20 induced by dynamic compression of human chondrocytes

Abstract: Objective Mechanical stimulation of cartilage affects tissue homeostasis and chondrocyte function. The chondrocyte phenotype is dependent on cell shape, which is largely determined by the actin cytoskeleton. Reorganization of the actin cytoskeleton results from Rho GTPase activation. The purpose of this study was to examine the roles of both actin and Rho in mechanotransduction in chondrocytes. Methods We embedded human articular chondrocytes in 2 × 6–mm agarose discs at 5 × 106 cells/ml and subjected the discs to unconfined dynamic compression at 0.5 Hz. By comparing samples with and without dynamic compression, we identified Rho activation according to the GTP-bound active RhoA measured in cell lysates. We identified rearrangements in filamentous actin structures using fluorescence-labeled phalloidin and confocal microscopy of fixed samples. We identified altered gene expression using TaqMan quantitative reverse transcription–polymerase chain reaction analysis. We tested for a requirement for Rho signaling by performing the dynamic compression in the presence of Rho kinase inhibitors. Results RhoA activation occurred within 5–10 minutes of dynamic compression. Rho kinase–dependent actin reorganization occurred within 20 minutes after application of dynamic compression and was apparent as “punctate” F-actin structures that were visible under confocal microscopy. We identified early-phase mechanoresponsive genes (CCL20 and inducible nitric oxide synthase) that were highly up-regulated within 1 hour of dynamic compression in a Rho kinase–dependent and actin-dependent manner. Conclusion Together, these results are the first demonstration that the Rho–Rho kinase pathway and actin cytoskeletal reorganization are required for changes in the expression of genes involved in human chondrocyte mechanotransduction.

A yoga-based exercise program to reduce the risk of falls in seniors: a pilot and feasibility study.

Abstract: We would like to thank the authors of “Response to a Proposal for an Integrative Medicine Curriculum.”1,2 We share their commitment to respectful collaboration among diverse health professionals, and are grateful for the thoughtful, detailed, constructive concerns and suggestions expressed in the paper. The Consortium of Academic Health Centers for Integrative Medicine (CAHCIM) (www.imconsortium.org) continues to evolve in its understanding and activities as it continues its basic commitment to patient-centered care. We welcome contributions such as this that will enhance future care and look forward to developing and strengthening our ACCAHC (Academic Consortium of Complementary & Alternative Health Care) and OCCIM (Oregon Collaborative for Complementary & Integrative Medicine) collaborations.

Hepatocytes Produce Interleukin-6a

Abstract: Interleukin-6 (IL-6) is now known to be the major hepatocyte stimulating factor, regulating the production of a wide spectrum of acute phase proteins (APP).' In previous studies, we have focused on the cytokines that stimulate synthesis of C1 esterase inhibitor (ClINH), which is the APP that controls activation of the complement and contact systems. Synthesis of ClINH in hepatocytes and hepatoma cells is stimulated by IFNy and IL-6. Interleukin1 (IL1 ) and tumor necrosis factor (TNF) do not have a direct effect on ClINH synthesis? However, when added together with IFNy, IL-1 increases ClINH synthesis, whereas it reduces the effect of IL-6 on synthesis of this APP. For other APP, it has been shown that IL-1 does not increase their synthesis in cultures of hepatoma cells or hepatocytes, whereas in uivo administration of IL-1 does raise plasma levels of these APP.) This in vivo effect of IL-1 is probably mediated by IL-6 because IL-1 induces IL-6 production in a variety of tissue^.\"^ In the present study, human hepatoma cells and primary human hepatocytes were examined as IL-1-responsive tissues for the ability to produce IL-6.

Interleukin‐6 (IL‐6) and Joint Tissues

Abstract: Inflammatory joint diseases are often associated with systemic changes, such as increased levels of acute phase proteins and hypergammaglobulinemia. Synoviocytes and chondrocytes probably play a central role through their location at the site of tissue destruction and through the secretion of proteases and other factors that contribute to the inflammatory process.' However, the cytokines that have been shown to be released by joint tissues cannot explain all systemic manifestations of these diseases. IL-6 is now known to be an important regulator of hepatocytes, of hematopoietic cells, and of B and T lymphocytes during host defense responses? We examined the regulation of IL-6 production by joint tissue cells and its presence in synovial fluids and we explored possible effects on joint tissues.