Gundula Schulze-Tanzil

Activation and dedifferentiation of chondrocytes: implications in cartilage injury and repair.

Cartilage injury remains a major challenge in orthopedic surgery due to the fact that articular cartilage has only a limited capacity for intrinsic healing. Cartilage impaction is followed by a post-traumatic inflammatory response. Chondrocytes and synoviocytes are activated to produce inflammatory mediators and degradative enzymes which can induce a progradient cartilage self-destruction finally leading to secondary osteoarthritis (OA). However, an anti-inflammatory compensatory response is also detectable in cartilage by up-regulation of anti-inflammatory cytokines, probably a temporary attempt by chondrocytes to restore cartilage homeostasis. Matrix-assisted autologous chondrocyte transplantation (MACT) is a suitable technique for improving the rate of repair of larger articular cartilage defects. For MACT, autologous chondrocytes were isolated from a cartilage biopsy of a non-load bearing joint area. This technique requires sufficient expansion of differentiated autologous chondrocytes, which were then seeded on suitable biodegradable three-dimensional (3D) matrices to preform an extracellular cartilage matrix (ECM) before implantation into the defect. Cell expansion is accompanied by chondrocyte dedifferentiation, whereby substantial changes occur at multiple levels of chondrocyte synthetic profiles: including the ECM, cell surface receptors and cytoskeletal proteins. Since these dedifferentiated chondrocytes produce a non-specific mechanically inferior ECM, they are not suitable for MACT. 3D cultures are means of inducing and maintaining chondrocyte (re)differentiation and to preform ECM. The combination of MACT with anabolic growth factors and anti-inflammatory strategies using anti-inflammatory mediators might be useful for stabilizing the differentiated chondrocyte phenotype, to support neocartilage formation and inhibit post-traumatic cartilage inflammation and hence, the development of secondary OA.

The Th17 cytokine IL‐22 induces IL‐20 production in keratinocytes: A novel immunological cascade with potential relevance in psoriasis

Psoriasis is a common chronic skin disease. Recent studies demonstrated that IL‐20 and IL‐22, cytokines produced by keratinocytes and T cells, respectively, both inhibit keratinocyte terminal differentiation and induce psoriasis‐like epidermis alterations. Here, we investigated the relationship between these mediators. Although IL‐20 was not able to regulate IL‐22 production, IL‐22 induced IL‐20 mRNA and protein in human keratinocytes. However, IL‐22 had only a minimal effect, if any, on IL‐19 and IL‐26. Cutaneous IL‐20 was also elevated in mice following IL‐22 application. Accordingly, some of IL‐22s effects on differentiation‐regulating genes were partially mediated by an endogenous, secreted protein and attenuated by anti‐IL‐20 Ab. Like IL‐22, IL‐17A and TNF‐α induced IL‐20 in keratinocytes, whereas IFN‐γ and IL‐20 itself did not. Furthermore, IL‐17A and TNF‐α individually strengthened the IL‐22‐induced IL‐20 production. In lesional skin of psoriasis patients, highly elevated IL‐20 levels strongly correlated with IL‐22, and to a lesser extent, with IL‐17A and TNF‐α. As previously shown for IL‐22, IL‐20 blood levels correlated with the disease severity, although with a lower significance. This study demonstrates that a T‐cell mediator induces a tissue cell mediator with similar effects to its own and therefore suggests the existence of a novel type of pathogenetic cascade.

Maturing dendritic cells are an important source of IL-29 and IL-20 that may cooperatively increase the innate immunity of keratinocytes.

IL‐19, IL‐20, IL‐22, IL‐24, IL‐26, IL‐28, and IL‐29 are new members of the IL‐10 interferon family. Monocytes are well‐known sources of IL‐19, IL‐20, and IL‐24. We demonstrated here that monocytes also expressed IL‐29, and monocyte differentiation into macrophages (Mφ) or dendritic cells (DCs) strongly changed their production capacity of these cytokines. Maturation of DCs with bacterial stimuli induced high expression of IL‐28/IL‐29 and IL‐20. Simulated T cell interaction and inflammatory cytokines induced IL‐29 and IL‐20 in maturing DCs, respectively. Compared with monocytes, DCs expressed only minimal IL‐19 levels and no IL‐24. The differentiation of monocytes into Mφ reduced their IL‐19 and terminated their IL‐20, IL‐24, and IL‐29 production capacity. Like monocytes, neither Mφ nor DCs expressed IL‐22 or IL‐26. The importance of maturing DCs as a source of IL‐28/IL‐29 was supported by the much higher mRNA levels of these mediators in maturing DCs compared with those in CMV‐infected fibroblasts, and the presence of IL‐28 in lymph nodes but not in liver of lipopolysaccharide‐injected mice. IL‐19, IL‐20, IL‐22, IL‐24, and IL‐26 do not seem to affect Mφ or DCs as deduced from the lack of corresponding receptor chains. The significance of IL‐20 and IL‐28/IL‐29 coexpression in maturing DCs may lie in the broadly amplified innate immunity in neighboring tissue cells like keratinocytes. In fact, IL‐20 induced the expression of antimicrobial proteins, whereas IL‐28/IL‐29 enhanced the expression of toll‐like receptors (TLRs) and the response to TLR ligands. However, the strongest response to TLR2 and TLR3 activation showed keratinocytes in the simultaneous presence of IL‐20 and IL‐29.

Simulated microgravity alters differentiation and increases apoptosis in human follicular thyroid carcinoma cells

This study focuses on the effects of simulated microgravity (0g) on the human follicular thyroid carcinoma cell line ML‐1. Cultured on a three‐dimensional clinostat, ML‐1 cells formed three‐dimensional MCTSs (MCTS diameter: 0.3±0.01 mm). After 24 and 48 h of clinorotation, the cells significantly decreased fT3 and fT4 secretion but up‐regulated the thyroid‐stimulating hormone‐receptor expression as well as the production of vimentin, vinculin, and extracellular matrix proteins (collagen I and III, laminin, fibronectin, chondroitin sulfate) compared with controls. Furthermore, ML‐1 cells grown on the clinostat showed elevated amounts of the apoptosis‐associated Fas protein, of p53, and of bax but showed reduced quantities of bcl‐2. In addition, signs of apoptosis became detectable, as assessed by terminal deoxynucleotidyl transferase‐mediated dUTP digoxigenin nick end labeling, 4′, 6‐diamidino‐2‐phenylindole staining, DNA laddering, and 85‐kDa apoptosis‐related cleavage fragments. These fragments resulted from enhanced 116‐kDa poly(ADP‐ribose)polymerase (PARP) activity and apoptosis. These observations suggest that clinorotation elevates intermediate filaments, cell adhesion molecules, and extracellular matrix proteins and simultaneously induces apoptosis in follicular thyroid cancer cells. In conclusion, our experiments could provide a regulatory basis for the finding that astronauts show low thyroid hormone levels after space flight, which may be explained by the increase of apoptosis in thyrocytes as a result of simulated 0g.

Apoptosis and the loss of chondrocyte survival signals contribute to articular cartilage degradation in osteoarthritis.

Apoptotic death of articular chondrocytes has been implicated in the pathogenesis of osteoarthritis (OA). Apoptotic pathways in chondrocytes are multi-faceted, although some cascades appear to play a greater in vivo role than others. Various catabolic processes are linked to apoptosis in OA cartilage, contributing to the reduction in cartilage integrity. Recent studies suggest that beta1-integrin mediated cell-matrix interactions provide survival signals for chondrocytes. The loss of such interactions and the inability to respond to IGF-1 stimulation may be partly responsible for the hypocellularity and matrix degradation that characterises OA. Here we have reviewed the literature in this area of cartilage cell biology in an effort to consolidate the existing information into a plausible hypothesis regarding the involvement of apoptosis in the pathogenesis of OA. Understanding of the interactions that promote chondrocyte apoptosis and cartilage hypocellularity is essential for developing appropriately targeted therapies for inhibition of chondrocyte apoptosis and the treatment of OA.

The role of pro‐inflammatory and immunoregulatory cytokines in tendon healing and rupture: new insights

Owing to limited self‐healing capacity, tendon ruptures and healing remain major orthopedic challenges. Increasing evidence suggests that post‐traumatic inflammatory responses, and hence, cytokines are involved in both cases, and also in tendon exercise and homeostasis. This review summarizes interrelations known between the cytokines interleukin (IL)‐1β, tumor necrosis factor (TNF)α, IL‐6 and vascular endothelial growth factor (VEGF) in tendon to assess their role in tendon damage and healing. Exogenic cytokine sources are blood‐derived leukocytes that immigrate in damaged tendon. Endogenous expression of IL‐1β, TNFα, IL‐6, IL‐10 and VEGF was demonstrated in tendon‐derived cells. As tendon is a highly mechanosensitive tissue, cytokine homeostasis and cell survival underlie an intimate balance between adequate biomechanical stimuli and disturbance through load deprivation and overload. Multiple interrelations between cytokines and tendon extracellular matrix (ECM) synthesis, catabolic mediators e.g. matrix‐degrading enzymes, inflammatory and angiogenic factors (COX‐2, PGE2, VEGF, NO) and cytoskeleton assembly are evident. Pro‐inflammatory cytokines affect ECM homeostasis, accelerate remodeling, amplify biomechanical adaptiveness and promote tenocyte apoptosis. This multifaceted interplay might both contribute to and interfere with healing. Much work must be undertaken to understand the particular interrelation of these inflammatory and regulatory mediators in ruptured tendon and healing, which has relevance for the development of novel immunoregulatory therapeutic strategies.

Effect of Pro-Inflammatory and Immunoregulatory Cytokines on Human Tenocytes

Tendon injury induces a local inflammatory response, characterized by the induction of pro‐inflammatory cytokines. The aim of the present study was to analyze the effects of TNFα, IL‐6 and IL‐10 on key parameters of tendon homeostasis. Cultured primary human tenocytes were treated with the recombinant cytokines IL‐6, IL‐10, TNFα, or combinations of TNFα with IL‐6 and IL‐10 (10 ng/mL, 6, 24 h). Expression of type I collagen, elastin, MMP‐1, TNFα, IL‐1β, IL‐6, IL‐10, and suppressors of cytokine signaling (SOCS1, 3) was analyzed with the use of RTD‐PCR, immunocytochemistry, and Western blot analysis. In response to TNFα, tenocytes reduced their type I collagen deposition but increased their elastin gene expression and highly upregulated their expression for MMP‐1, pro‐inflammatory (TNFα, IL‐1β) and immunoregulatory (IL‐6, IL‐10) cytokines. TNFα stimulation augmented SOCS1, whereas SOCS3 expression in tenocytes was also induced by IL‐6. The treatment of tenocytes with IL‐6 and IL‐10 had no effect on cytokine expression. Neither IL‐6 nor IL‐10 modulated the observed effects of TNFα significantly. These results indicate that TNFα strongly activates the tenocytes to amplify their own TNFα expression and, subsequently, that of other regulatory cytokines and matrix degrading enzymes. However, the impact of IL‐6 and IL‐10 on tenocytes remains unclear.

Effects of Curcumin (Diferuloylmethane) on Nuclear Factor κB Signaling in Interleukin‐1β‐Stimulated Chondrocytes

Abstract: Curcumin (diferuloylmethane) is a nontoxic dietary pigment in tumeric and curry and a potent inhibitor of the common transcription factor Nuclear Factor κB (NF‐κB) in several cell types. It is well established that some of the catabolic effects of the proinflammatory cytokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐α in osteoarthritis are regulated by the activation of NF‐κB. Therefore, the aim of this study was to determine whether curcumin modifies the catabolic response of chondrocytes to IL‐1β. Human articular chondrocytes were prestimulated with 10 ng/mL IL‐1β for 0, 4, 8, 12, or 24 h and then cotreated with 50 μM curcumin for 0, 12, 24, 36, or 48 h. Synthesis of the cartilage‐specific collagen type II and matrix‐degrading enzyme matrix metalloproteinase‐3 (MMP‐3) was investigated in chondrocytes by Western blot analysis. Activation and nuclear translocation of NF‐κB were observed by immunofluorescence microscopy. IL‐1β induced a decrease in collagen type II and upregulation of MMP‐3 in a time‐dependent manner. Upregulation of MMP‐3 was inhibited by curcumin in a time‐dependent manner. In addition, IL‐1β‐induced a decrease in type II collagen, which was relieved by curcumin treatment. In response to IL‐1β, NF‐κB translocated to the nucleus, but translocation was inhibited by curcumin, as revealed by immunofluorescence microscopy. Taken together, these results confirmed an IL‐1β‐mediated upregulation of proinflammatory MMP‐3 in chondrocytes via an NF‐κB activation mechanism. Curcumin protected chondrocytes from the catabolic effects of IL‐1β, such as MMP‐3 upregulation, and interestingly also relieved cytokine‐induced suppression of matrix protein synthesis. Therefore, curcumin antagonizes crucial catabolic effects of IL‐1β signaling that are known to contribute to the pathogenesis of osteoarthritis.

Early onset of chondroitin sulfate and osteopontin expression in angiotensin ii-dependent left ventricular hypertrophy

BACKGROUND Chondroitin sulfate proteoglycan (CSPG) is expressed during embryonic heart development and osteopontin (OPN) is an important mediator of the profibrotic effects of angiotensin II (Ang II). The objective of this study was to analyze extracellular matrix protein (ECMP) expression in Ang II-dependent left ventricular (LV) hypertrophy (LVH), LV dysfunction, and to investigate right ventricular changes. METHODS We used the hypertensive transgenic rat line TGR(mRen2)27 (Ren2), which provides a well-established model of Ang II-driven cardiac remodeling and progressive LV dysfunction and compared young Ren2 rats at the age of 10 weeks with normotensive Sprague-Dawley (SD) rats (n = 15, each group). RESULTS Systolic blood pressure and LV weight were elevated in Ren2 compared to SD rats (P < .001). Left ventricular end-diastolic pressure was not altered in Ren2, but +dP/dt(max) and -dP/dt(max) were decreased in Ren2 compared to SD rats (P < .01). Cardiomyocyte widths, interstitial and perivascular fibrosis were increased in left and right ventricles of Ren2 in comparison to SD rats (P < .05). The LV mRNA expression of atrial natriuretic factor, OPN, and collagen I were increased in Ren2 as compared to SD rats (P < .05, respectively). The LV CSPG, collagen I, collagen III, fibronectin, laminin, and OPN contents were elevated in Ren2 compared to SD rats as measured by image analysis and Western blotting (P < .01). CONCLUSIONS Reactivated expression of CSPG in the adult heart may be an important component of LV ECMP remodeling in LVH. Elevated cardiac OPN expression could mediate the alterations in LV ECMP pattern in Ang II-dependent LVH, thus contributing to the development of contractile dysfunction in young Ren2 rats.

Extracellular matrix expression of human tenocytes in three-dimensional air-liquid and PLGA cultures compared with tendon tissue: implications for tendon tissue engineering.

Tenocyte transplantation may prove to be an approach to support healing of tendon defects. Cell–cell and cell–matrix contacts within three‐dimensional (3D) cultures may prevent tenocyte dedifferentiation observed in monolayer (2D) culture. The present study compares both neotissue formation and tenocyte extracellular matrix (ECM) expression in 2D and 3D cultures directly with that of native tendon, in order to determine optimal conditions for tendon tissue engineering. Primary human tenocytes were embedded in poly[lactic‐co‐glycolic‐acid] (PLGA)‐scaffolds and high‐density cultures. Neotissue formation was examined by hematoxyline–eosine (H&E) and immunofluorescence staining. Gene expression of ECM proteins and vascular endothelial growth factor (VEGF) was compared at days 0 (2D), 14, and 28 in 3D cultures and tendon. Histomorphology of 3D culture showed tendon‐like tissue as tenocyte cell nuclei became more elongated and ECM accumulated. Type I collagen gene expression was higher in 2D culture than in tendon and decreased in 4‐week‐old 3D cultures, whereas type III collagen was only elevated in high‐density culture compared with tendon. Decorin and COMP were reduced in 2D and increased in 3D culture almost to ex vivo level. These results suggest that the 3D high‐density or biodegradable scaffolds cultures encourage the differentiation of expanded monolayer tenocytes in vitro to tendon‐like tissue.