Biochemische Wirkungen von organischen Metallverbindungen auf in vitro Zellsysteme: Untersuchungen zur zellulären Signaltransduktion und Apoptose

In der vorliegenden Arbeit wurden intrazelluläre Signaltransduktionsmechanismen unter dem Einfluß von organischen Zinnverbindungen untersucht. Als Modellsubstanz wurde hier die für die Umwelt relevanteste Verbindung Tributylzinn (TBT) verwendet. Die TBT-induzierte Arachidonsäurefreisetzung wurde untersucht und der hierfür verantwortliche Mechanismus aufgeklärt: TBT induziert über die Aktivierung der zytosolischen Phospholipase A2 (cPLA2) die Freisetzung von Arachidonsäure. Anschließend wurde die Reaktionsfolge, die zur Aktivierung der cPLA2 führte, näher charakterisiert. Die Behandlung mit TBT induzierte über eine sehr schnelle Aktivierung der MAP Kinase Kaskade die Phosphorylierung der cPLA2 und schließlich die Freisetzung von Arachidonsäure. Diese Ergebnisse waren bei HL-60 Zellen, P388D1 Zellen und HeLa Zellen identisch. TBT stimulierte außerdem die Aktivität der c-Jun NH2-terminalen Kinase (JNK). In dieser Arbeit wurde damit erstmals die Aktivierung verschiedener Proteinkinasen durch organische Metallverbindungen nachgewiesen. Damit kommt zur bereits bekannten Störung der intrazellulären Calciumhomöostase durch organische Metallverbindungen ein neuer Wirkungsmechanismus hinzu, der die calcium-abhängigen Effekte noch verstärken kann. Versuche mit Calciumchelatoren bewiesen, daß für die TBT-induzierte Aktivierung der Proteinkinasen eine intrazelluläre Calciumerhöhung zwar essentiell aber allein nicht ausreichend ist. Im zweiten Teil der Arbeit wurden Untersuchungen zur induzierten Apoptose durchgeführt. Hierbei stand die Untersuchung spezifischer morphologischer Veränderungen, der Fragmentierung der DNA und proteolytischer Prozesse im Vordergrund. Mit verschiedenen mikroskopischen und biochemischen Techniken wurde der organometall-induzierte Zelltod eindeutig als Apoptose charakterisiert. Die Apoptose wurde innerhalb weniger Stunden induziert, und auch hier waren die Effekte von TBT vom untersuchten Zelltyp unabhängig. Die Aktivierung von Caspasen und Endonukleasen ist verantwortlich für die spezifischen Abbauprozesse nach Behandlung mit TBT. Der Beweis hierfür wurde durch die Vorbehandlung mit Capase Inhibitoren erbracht. Dies führte zur Aufhebung der metall-induzierten Effekte auf allen Ebenen. Die Inkubation mit apoptose-induzierenden Konzentrationen von TBT führte zur Aktivierung von Kinasen aus der MAP Kinase Familie. Durch Verwendung von spezifischen Kinase Inhibitoren wurde ein Zusammenhang zwischen der Aktivierung von Proteinkinasen und der Induktion der Apoptose hergestellt. Die im Rahmen dieser Arbeit nachgewiesene Induktion der Apoptose durch Organometalle könnte für die bereits beschriebenen immunotoxischen und neurotoxischen aber auch die antiproliferativen Effekte einiger Verbindungen verantwortlich sein. Die hier beschriebene Vielfalt der durch organische Metallverbindungen induzierten tiefgreifenden Reaktionen, führte im Hinblick auf die bereits nachgewiesenen Konzentrationen in der Umwelt zu dem Schluß, daß die weitere Verwendung und Zulassung dieser Verbindungsklasse in Zukunft äußerst kritisch überdacht werden sollte

Osteoarthritis: Novel Molecular Mechanisms Increase Our Understanding of the Disease Pathology

Although osteoarthritis (OA) is the most common musculoskeletal condition that causes significant health and social problems worldwide, its exact etiology is still unclear. With an aging and increasingly obese population, OA is becoming even more prevalent than in previous decades. Up to 35% of the world’s population over 60 years of age suffers from symptomatic (painful, disabling) OA. The disease poses a tremendous economic burden on the health-care system and society for diagnosis, treatment, sick leave, rehabilitation, and early retirement. Most patients also experience sleep disturbances, reduced capability for exercising, lifting, and walking and are less capable of working, and maintaining an independent lifestyle. For patients, the major problem is disability, resulting from joint tissue destruction and pain. So far, there is no therapy available that effectively arrests structural deterioration of cartilage and bone or is able to successfully reverse any of the existing structural defects. Here, we elucidate novel concepts and hypotheses regarding disease progression and pathology, which are relevant for understanding underlying the molecular mechanisms as a prerequisite for future therapeutic approaches. Emphasis is placed on topographical modeling of the disease, the role of proteases and cytokines in OA, and the impact of the peripheral nervous system and its neuropeptides

β1 Integrins Mediate Attachment of Mesenchymal Stem Cells to Cartilage Lesions

Mesenchymal stem cells (MSC) may have great potential for cell-based therapies of osteoarthritis. However, after injection in the joint, only few cells adhere to defective articular cartilage and contribute to cartilage regeneration. Little is known about the molecular mechanisms of MSC attachment to defective articular cartilage. Here, we developed an ex vivo attachment system, using rat osteochondral explants with artificially created full-thickness cartilage defects in combination with genetically labeled MSC isolated from bone marrow of human placental alkaline phosphatase transgenic rats. Binding of MSC to full-thickness cartilage lesions was improved by serum, but not hyaluronic acid, and was dependent on the presence of divalent cations. Additional in vitro tests showed that rat MSC attach, in a divalent cation-dependent manner, to collagen I, collagen II, and fibronectin, but not to collagen XXII or cartilage oligomeric matrix protein (COMP). RGD peptides partially blocked the adhesion of MSC to fibronectin in vitro and to cartilage lesions ex vivo. Furthermore, the attachment of MSC to collagen I and II in vitro and to cartilage lesions ex vivo was almost completely abolished in the presence of a β1 integrin blocking antibody. In conclusion, our data suggest that attachment of MSC to ex vivo full-thickness cartilage lesions is almost entirely β1 integrin-mediated, whereby both RGD- and collagen-binding integrins are involved. These findings suggest a key role of integrins during MSC attachment to defective cartilage and may pave the way for improved MSC-based therapies in the future

Comp and tsp-4: Functional roles in articular cartilage and relevance in osteoarthritis

Osteoarthritis (OA) is a slow-progressing joint disease, leading to the degradation and remodeling of the cartilage extracellular matrix (ECM). The usually quiescent chondrocytes become reactivated and accumulate in cell clusters, become hypertrophic, and intensively produce not only degrading enzymes, but also ECM proteins, like the cartilage oligomeric matrix protein (COMP) and thrombospondin-4 (TSP-4). To date, the functional roles of these newly synthesized proteins in articular cartilage are still elusive. Therefore, we analyzed the involvement of both proteins in OA specific processes in in vitro studies, using porcine chondrocytes, isolated from femoral condyles. The effect of COMP and TSP-4 on chondrocyte migration was investigated in transwell assays and their potential to modulate the chondrocyte phenotype, protein synthesis and matrix formation by immunofluorescence staining and immunoblot. Our results demonstrate that COMP could attract chondrocytes and may contribute to a repopulation of damaged cartilage areas, while TSP-4 did not affect this process. In contrast, both proteins similarly prom

Comp and tsp-4: Functional roles in articular cartilage and relevance in osteoarthritis

Osteoarthritis (OA) is a slow-progressing joint disease, leading to the degradation and remodeling of the cartilage extracellular matrix (ECM). The usually quiescent chondrocytes become reactivated and accumulate in cell clusters, become hypertrophic, and intensively produce not only degrading enzymes, but also ECM proteins, like the cartilage oligomeric matrix protein (COMP) and thrombospondin-4 (TSP-4). To date, the functional roles of these newly synthesized proteins in articular cartilage are still elusive. Therefore, we analyzed the involvement of both proteins in OA specific processes in in vitro studies, using porcine chondrocytes, isolated from femoral condyles. The effect of COMP and TSP-4 on chondrocyte migration was investigated in transwell assays and their potential to modulate the chondrocyte phenotype, protein synthesis and matrix formation by immunofluorescence staining and immunoblot. Our results demonstrate that COMP could attract chondrocytes and may contribute to a repopulation of damaged cartilage areas, while TSP-4 did not affect this process. In contrast, both proteins similarly prom

Uromodulin is expressed in renal primary cilia and UMOD mutations result in decreased ciliary uromodulin expression

Uromodulin (UMOD) mutations are responsible for three autosomal dominant tubulo-interstitial nephropathies including medullary cystic kidney disease type 2 (MCKD2), familial juvenile hyperuricemic nephropathy and glomerulocystic kidney disease. Symptoms include renal salt wasting, hyperuricemia, gout, hypertension and end-stage renal disease. MCKD is part of the ‘nephronophthisis-MCKD complex', a group of cystic kidney diseases. Both disorders have an indistinguishable histology and renal cysts are observed in either. For most genes mutated in cystic kidney disease, their proteins are expressed in the primary cilia/basal body complex. We identified seven novel UMOD mutations and were interested if UMOD protein was expressed in the primary renal cilia of human renal biopsies and if mutant UMOD would show a different expression pattern compared with that seen in control individuals. We demonstrate that UMOD is expressed in the primary cilia of renal tubules, using immunofluorescent studies in human kidney biopsy samples. The number of UMOD-positive primary cilia in UMOD patients is significantly decreased when compared with control samples. Additional immunofluorescence studies confirm ciliary expression of UMOD in cell culture. Ciliary expression of UMOD is also confirmed by electron microscopy. UMOD localization at the mitotic spindle poles and colocalization with other ciliary proteins such as nephrocystin-1 and kinesin family member 3A is demonstrated. Our data add UMOD to the group of proteins expressed in primary cilia, where mutations of the gene lead to cystic kidney diseas

β2-Adrenoceptor Deficiency Results in Increased Calcified Cartilage Thickness and Subchondral Bone Remodeling in Murine Experimental Osteoarthritis

Purpose: Recent studies demonstrated a contribution of adrenoceptors (ARs) to osteoarthritis (OA) pathogenesis. Several AR subtypes are expressed in joint tissues and the β2-AR subtype seems to play a major role during OA progression. However, the importance of β2-AR has not yet been investigated in knee OA. Therefore, we examined the development of knee OA in β2-AR-deficient (Adrb2-/- ) mice after surgical OA induction. Methods: OA was induced by destabilization of the medial meniscus (DMM) in male wildtype (WT) and Adrb2-/- mice. Cartilage degeneration and synovial inflammation were evaluated by histological scoring. Subchondral bone remodeling was analyzed using micro-CT. Osteoblast (alkaline phosphatase - ALP) and osteoclast (cathepsin K - CatK) activity were analyzed by immunostainings. To evaluate β2-AR deficiency-associated effects, body weight, sympathetic tone (splenic norepinephrine (NE) via HPLC) and serum leptin levels (ELISA) were determined. Expression of the second major AR, the α2-AR, was analyzed in joint tissues by immunostaining. Results: WT and Adrb2-/- DMM mice developed comparable changes in cartilage degeneration and synovial inflammation. Adrb2-/- DMM mice displayed elevated calcified cartilage and subchondral bone plate thickness as well as increased epiphyseal BV/TV compared to WTs, while there were no significant differences in Sham animals. In the subchondral bone of Adrb2-/- mice, osteoblasts activity increased and osteoclast activity deceased. Adrb2-/- mice had significantly higher body weight and fat mass compared to WT mice. Serum leptin levels increased in Adrb2-/- DMM compared to WT DMM without any difference between the respective Shams. There was no difference in the development of meniscal ossicles and osteophytes or in the subarticular trabecular microstructure between Adrb2-/- and WT DMM as well as Adrb2-/- and WT Sham mice. Number of α2-AR-positive cells was lower in Adrb2-/- than in WT mice in all analyzed tissues and decreased in both Adrb2-/- and WT over time. Conclusion: We propose that the increased bone mass in Adrb2-/- DMM mice was not only due to β2-AR deficiency but to a synergistic effect of OA and elevated leptin concentrations. Taken together, β2-AR plays a major role in OA-related subchondral bone remodeling and is thus an attractive target for the exploration of novel therapeutic avenues

Genetic mouse models for the functional analysis of the perifibrillar components collagen IX, COMP and matrilin-3: Implications for growth cartilage differentiation and endochondral ossification

The mutual interaction of the two supramolecular compartments, the fibrillar and extrafibrillar matrix is a prerequisite for stability and integrity of the cartilage extracellular matrix. The fibrillar periphery, composed of collagen IX, matrilins and cartilage oligomeric matrix protein (COMP) among other components, constitutes the interface which mediates interactions between the two compartments. Mutations in these peripheral macromolecules cause a broad spectrum of skeletal conditions such as pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED), which severely affect the organization and integrity of the cartilage growth matrix in humans. Transgenic and knockout mouse models for collagen IX, matrilin-3 and COMP and combinations thereof display cartilage abnormalities and pathologies of varying severity. Absence of collagen IX appears to cause the most severe growth plate phenotype with a profoundly disturbed morphological organization affecting size and shape of the long bones. Notably, similar growth plate phenotypes, including irregularities in the proteoglycan content, hypocellular central regions, disorganized proliferation columns with atypically shaped and oriented chondrocytes and alterations in the hypertrophic zone are observed in transgenic mice lacking other macromolecules or carrying mutations therein. These include collagens II and XI, integrin subunits, integrin linked kinase (ILK), HIF-1α, VEGFα and the tumor suppressor PTEN. Notably, mutations in ciliar proteins such as Kif3α, polaris or Smo/Gli severely affect the ability of chondrocytes to move and to become arranged in columns. Absence or mutational changes of a variety of different, non-related cartilage macromolecules apparently cause similar pathologies and abnormalities of the growth cartilage, suggesting a limited number of underlying molecular mechanism

Editorial: Bone and Cartilage Regeneration With Extracellular Vesicles

none3siThe primary aim of regenerative medicine is to stimulate tissue healing. Despite the great promises raised by the use of stem/progenitor cells for skeletal tissue regeneration, cell tracking analysis has revealed that transplanted cells do not commonly become part of the injured tissue (Pittenger et al., 2019). The role of the stem cell-derived secretome is becoming increasingly intriguing due to its ability to stimulate endogenous regenerative processes. Therefore, the so-called “paracrine hypothesis” has taken hold (Gnecchi et al., 2016). Extracellular vesicles (EVs) are important components of the cell secretome, representing promising tools for the delivery of biologically active molecules which can be used for therapeutic purposes (Alcaraz et al., 2019). EVs are bilayer membrane fragments released by almost any cell type upon activation or death. Two major types of EVs are usually distinguished: exosomes, formed from the endosomal cell compartment, and microvesicles, produced by the direct extrusion from the cell plasma membrane (Doyle and Wang, 2019). Nowadays, the effects specifically exerted by one EV subpopulation over another are still unclear. This is in part due to their overlapping size and variable cargos, which does not allow a precise discrimination between them. EVs contain proteins, lipids, and nucleic acids and have the potential to activate not only complementary, pro-regenerative signaling pathways in the same responder cells, but also to stimulate multiple target cell populations and tissues. This property could make them an efficient therapeutic vehicle for bone and cartilage regenerative medicine. However, there are many concerns that should be addressed, such as the development of strategies to obtain sufficient amounts of EVs, the identification and characterization of the optimal donor cell source, the necessity to develop ideal scaffolds to be used as depots for controlled release of EVs, as well as the need to better understand the mechanisms underlying bone/cartilage formation after EV treatment.openRoberta Tasso, Susanne Grässel, Frank ZauckeTasso, Roberta; Grässel, Susanne; Zaucke, Fran

Insulin-like growth factor 1 receptor (IGF1R) signaling regulates osterix expression and cartilage matrix mineralization during endochondral ossification

Insulin-like growth factor I receptor (IGF1R) signaling is important for bone formation via endochondral ossification. Igf1r deficient mice show proportional dwarfism and alterations in chondrocyte proliferation, hypertrophy and apoptosis within the growth plate. In addition, gene ablation in mouse demonstrated that IGF1R signaling is important for osteoblast mediated bone mineralization. However, the mineralization in the terminal hypertrophic zone of the growth plate is also an essential step in endochondral ossification preceding bone formation. Therefore, we analyzed the influence of IGF1R signaling on this process by using mice with a specific deletion of Igf1r in chondrocytes. Studies in embryonic metatarsal explant cultures showed that mineralization of the terminal hypertrophic zone was strongly reduced when IGF1R signaling was lacking. This decreased mineralization may in part result from the delay in hypertrophic differentiation in the Igf1r deficient metatarsals. However, mineralization was impaired even stronger than hypertrophy, suggesting a mineralization promoting effect of IGF signaling that is independent of hypertrophic differentiation. We found a markedly decreased osterix expression suggesting that osterix is a downstream target of IGF1R in chondrocytes. MMP13 expression was strongly reduced in metatarsals lacking the IGF1R while alkaline phosphatase expression and activity were less affected. We conclude that endogenous IGF1R signaling is important for growth plate matrix remodeling and calcification leading to bone formation and suggest that regulation of osterix expression and its downstream target MMP13 are part of the underlying mechanism. (c) 2015 Elsevier Inc. All rights reserved