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Purpose: Recent studies demonstrated a contribution of adrenoceptors (ARs) to osteoarthritis (OA) pathogenesis. Several AR subtypes are expressed in joint tissues and the ��2-AR subtype seems to play a major role during OA progression. However, the importance of ��2-AR has not yet been investigated in knee OA. Therefore, we examined the development of knee OA in ��2-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 ��2-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 ��2-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 ��2-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 ��2-AR deficiency but to a synergistic effect of OA and elevated leptin concentrations. Taken together, ��2-AR plays a major role in OA-related subchondral bone remodeling and is thus an attractive target for the exploration of novel therapeutic avenues.

Selection of appropriate housekeeping genes is essential for the validity of data normalization in reverse transcription quantitative PCR (RT-qPCR). Synovial fibroblasts (SF) play a mediating role in the development and progression of osteoarthritis (OA) pathogenesis, but there is no information on reliable housekeeping genes available. Therefore the goal of this study was to identify a set of reliable housekeeping genes suitable for studies of mechanical loading on SF from healthy and OA patients. Nine genes were evaluated towards expression stability and ranked according their relative stability determined by four different mathematical procedures (geNorm, NormFinder, BestKeeper and comparative ΔCq). We observed that RPLP0 (ribosomal protein, large, P0) and EEF1A1 (eukaryotic translation elongation factor 1 alpha 1) turned out to be the genes with the most stable expression in SF from non-OA or OA patients treated with or without mechanical loading. According to geNorm two genes are sufficient for normalization throughout. Expression of one tested target gene varied considerably, if normalized to different candidate housekeeping genes. Our study provides a tool for accurate and valid housekeeping gene selection in gene expression experiments on SF from healthy and OA patients with and without mechanical loading in consistent with the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and additionally demonstrates the impact of proper housekeeping gene selection on the expression of the gene of interest.

Osteoarthritis (OA) affects the integrity of the entire joint including the synovium. The most abundant cells in the synovium are fibroblasts (SF). Excessive mechanical loading might contribute to OA pathogenesis. Here, we investigate the effects of mechanical loading on SF derived from non-OA (N-SF) and OA patients (OA-SF). We treated N-SF and OA-SF with or without mechanical loading for 48h after 24h of preincubation. Then we assessed gene and protein expression of proinflammatory factors (TNF&alpha, COX-2, PG-E2, IL-6), extracellular matrix (ECM) components (COL1, FN1) and glycosaminoglycans (GAGs) via RT-qPCR, ELISA, DMMB assay and HPLC. Mechanical loading significantly increased TNF&alpha, and PG-E2 secretion by N-SF and OA-SF, whereas in OA-SF IL-6 secretion was reduced. COL1 and FN1 secretion were downregulated in N-SF during loading. OA-SF secreted less COL1 compared to N-SF under control conditions. In contrast, OA-SF in general expressed more FN1. GAG synthesis was upregulated in N-SF, but not in OA-SF during loading with OA-SF displaying a higher charge density than N-SF. Mechanical loading enhanced proinflammatory factor expression and GAG synthesis and decreased secretion of ECM components in N-SFs, indicating a contributing role of SF to OA development.

Joint tissues like synovium, articular cartilage, meniscus and subchondral bone, are targets for neuropeptides. Resident cells of these tissues express receptors for various neuroendocrine-derived peptides including proopiomelanocortin (POMC)-derived peptides, i.e., α-melanocyte-stimulating hormone (α-MSH), adrenocorticotropin (ACTH) and β-endorphin (β-ED), and sympathetic neuropeptides like vasoactive intestinal peptide (VIP) and neuropeptide y (NPY). Melanocortins attained particular attention due to their immunomodulatory and anti-inflammatory effects in several tissues and organs. In particular, α-MSH, ACTH and specific melanocortin-receptor (MCR) agonists appear to have promising anti-inflammatory actions demonstrated in animal models of experimentally induced arthritis and osteoarthritis (OA). Sympathetic neuropeptides have obtained increasing attention as they have crucial trophic effects that are critical for joint tissue and bone homeostasis. VIP and NPY are implicated in direct and indirect activation of several anabolic signaling pathways in bone and synovial cells. Additionally, pituitary adenylate cyclase-activating polypeptide (PACAP) proved to be chondroprotective and, thus, might be a novel target in OA. Taken together, it appears more and more likely that the anabolic effects of these neuroendocrine peptides or their respective receptor agonists/antagonists may be exploited for the treatment of patients with inflammatory and degenerative joint diseases in the future.

Makrophagen sind Vorläuferzellen von Osteoklasten, deren übermäßige Aktivierung in Rheumatoider Arthritis (RA) zur Erosion des gelenknahen Knochens beiträgt. Neurotransmitter des sympathischen Nervensystems wie Noradrenalin (NA), Acetylcholin (ACh) und vasoaktives intestinales Peptid (VIP) wirken als Modulatoren der Arthritis. Arthritis induziert Veränderungen in der lokalen synovialen Innervierung, die ein lokal verändertes Neurotransmitter-Mikromilieu erzeugen. Knochen- und Periostzellen können Faktoren exprimieren, die in der Lage sind, eine Veränderung des katecholaminergen Phänotyps von Nervenfasern in einen cholinerg / peptidergen (VIP) Phänotyp zu induzieren und somit eine neue regulatorische Neurotransmitterumgebung zu schaffen. In einem DA-Rattenmodell der Kollagen Typ II-induzierten Arthritis (CIA) sollte daher der Einfluss der Neurotransmitter ACh, NA und VIP auf zelluläre und metabolische Eigenschaften von Knochenmark-Makrophagen (KMM) und deren Osteoklastogenesekapazität in verschiedenen Arthritisstadien untersucht werden. Sowohl KMM als auch Osteoklasten exprimieren Rezeptoren für ACh, NA und VIP und können somit auf Neurotransmitterreize reagieren. Die Genexpressionsrate verschiedener Rezeptorgene wird durch CIA in Osteoklastenkulturen größtenteils gehemmt, aber auf Proteinebene wurden nach qualitativer Auswertung keine Unterschiede in der Rezeptorexpression und -lokalisation der Adrenozeptoren alpha1D, alpha2B und beta2, dem muskarinergen ACh Rezeptor M5 sowie dem alternativen VIP-Rezeptor PACAP-Rezeptor 1 festgestellt. Bemerkenswert ist, dass die Expression muskarinerger ACh Rezeptoren zuvor noch nicht auf Osteoklasten nachgewiesen wurde. Einflüsse von CIA auf die intrazelluläre Signalkaskade können aber nicht ausgeschlossen werden und sind in dieser Arbeit nicht analysiert worden. Auffällig war, dass nach Isolierung über Plastikadhärenz, die Anzahl der KMM aus akut arthritischen Ratten signifikant kleiner war im Vergleich zu Kontrollratten. Parallel war dagegen der Anteil der myeloiden Zellpopulation im nativen Knochenmark durch akute CIA stark erhöht, was den erhöhten Bedarf an Immunzellen in Entzündungsreaktionen widerspiegelt. Als Ursache der reduzierten KMM-Anzahl nach Isolierung konnte eine starke Hemmung der Plastikadhäsion von KMM aus arthritischen Ratten beobachtet werden. Parallel dazu war die Adhäsion an Komponenten der extrazellulären Matrix wie Kollagen Typ I und Fibronektin in der chronischen Phase der Arthritis gehemmt, die in vivo zu einer Inhibition der Migration von KMM im Gelenk führen kann und somit das Aufflammen einer erneuten Entzündung verhindern könnte. Die Adhäsion an Kollagen Typ I und Laminin wurde in KMM aus Kontrolltieren durch NA gehemmt, während CIA-KMM keine Reaktion zeigten. Die Plastikadhäsion wurde dagegen durch NA in Kontroll-KMM verstärkt, während CIA-KMM keine Reaktion nach NA-Stimulation zeigten, dafür aber eine verstärkte Plastikadhärenz nach VIP-Stimulation. Dadurch konnte deutlich gezeigt werden, dass CIA die NA-gesteuerte Adhäsion moduliert und dass zumindest bezüglich der Adhärenz an Plastik, VIP die Anergie der CIA-KMM gegenüber NA-Stimulation kompensieren kann. Des weiteren wurden schon früh nach der Immunisierung Mechanismen induziert, die zu einer Hemmung der Proliferationskapazität von KMM hauptsächlich in CIA-Phasen mit geringer oder keiner Entzündung führen. Im akuten CIA-Stadium wird der hemmende Einfluss durch unbekannte Mechanismen aufgehoben und führt, unter Berücksichtigung der reduzierten Adhäsion, eher zu einer verstärkten Proliferation von KMM. Anti-proliferative Effekte von NA, ACh und VIP auf Kontroll-KMM wurden durch CIA moduliert und teilweise gehemmt. Bezüglich der Caspase 3/7 Aktivität induziert CIA mit dem Auftreten erster Symptome einen Switch in der Reaktivität nach Stimulation mit ACh, NA und VIP, der protektiv auf KMM wirkt. Damit haben wir deutliche Hinweise auf eine CIA-induzierte Regulation der Neurotransmitter-Reaktivität von KMM erhalten, die abhängig von der jeweiligen CIA-Phase ist und zelluläre Prozesse wie Adhäsion, Apoptose und Proliferation betrifft. Ein weiterer wichtiger Punkt, der in dieser Arbeit untersucht wurde, ist die Osteoklastogenese von KMM in verschiedenen Stadien experimenteller Arthritis und der gleichzeitige Einfluss der Neurotransmitter ACh, NA und VIP. CIA verändert die Osteoklastogenese von KMM nicht auf der Differenzierungs-, aber auf der Aktivitätsebene, wie durch eine Hemmung der Kathepsin K-Aktivität im Kulturüberstand von Osteoklasten gezeigt wurde. Eine Hemmung der Produktion oder Aktivität von Kathepsin K wäre als eine Gegenmaßnahme des Organismus zu verstehen, die einen weiteren Abbau der Knochenmatrix verhindern oder verlangsamen könnte. Eine verstärkte Caspase 3/7-Aktivität nach M-CSF/Rankl-Deprivation in Osteoklastenkulturen aus CIA-Ratten, könnte ebenfalls eine regulatorische Maßnahme des Organismus darstellen, um der großen Anzahl von Osteoklasten in den gelenknahen Regionen entgegenzuwirken. Die Genexpressionsrate verschiedener Differenzierungs- und Aktivierungsmarker wurde durch CIA phasenabhängig gehemmt, scheint sich aber nicht auf Proteinebene zu manifestieren. Zusätzlich dazu konnten wir einen starken inhibierenden Effekt von NA und VIP auf die Osteoklastenaktivität beobachten, der vor allem auf einer Hemmung der Kathepsin K-Aktivität beruhte und in vivo protektiv für die Knochenmatrix wäre. Vermittelt wird dieser Effekt zumindest teilweise über den cAMP-Signalweg, wie wir durch entsprechende Experimente mit einem Adenylatzyklase-Agonisten nachweisen konnten. Konträr dazu wurde durch ACh die Kathepsin K-Aktivität und die Matrixresorption verstärkt, sowie die Anzahl der gebildeten Osteoklasten erhöht. Die im Gegensatz dazu beobachtete dosisabhängige Hemmung der Osteoklastogenese und verstärkte Caspase 3/7-Aktivität nach Stimulation mit NA/VIP unterstützt den Befund, dass das cholinerge und adrenerge Nervensystem Gegenspieler in der in vitro-Osteoklastogenese und womöglich auch in der CIA-Pathogenese darstellen. Die Modulation der Sensitivität gegenüber der Stimulation mit ACh, NA und VIP durch CIA ermöglicht Osteoklasten eine Adaptation an Veränderungen in der lokalen Neurotransmitterzusammensetzung in den verschiedenen arthritischen Erkrankungsphasen., Macrophages are key players in Rheumatoid Arthritis (RA) and provide the source of precursor cells for osteoclasts. Osteoclasts are bone resorbing cells responsible for the destruction of periarticular bone in rheumatoid lesions. Neurotransmitters of the sympathetic nervous system like acetylcholine (ACh), noradrenaline (NA) and vasoactive intestinal peptide (VIP) act as modulators of RA. Changes in local innervation induced by arthritis change local neurotransmitter composition and concentration and provide a novel regulatory environment for osteoclasts and their respective precursors, bone marrow-derived macrophages (BMM). This thesis therefore intended to analyze the influence of ACh, NA and VIP on intrinsic metabolic properties of BMM and their osteoclastogenic capacity in the context of various stages of a collagen type II-induced arthritis (CIA) model in DA rats. Both, osteoclasts and BMM express receptors for and therefore respond to ACh, NA and VIP stimulation. We detected that gene expression is affected by CIA but the effects do not manifest on protein level as we have shown for adrenoceptors alpha1D, alpha2B, beta2, muscarinic ACh receptor M5 and the alternative VIP receptor PACAP receptor 1. Nevertheless, an influence of CIA on intracellular signaling cascades of these receptors cannot be excluded. One striking observation of this thesis was the reduced number of isolated BMM after separation from native bone marrow via plastic adherence at the acute CIA stage in comparison to control BMM numbers. This observation was in strong contrast to enhanced numbers of myloid cells (belonging to the macrophage proportion) in native bone marrow preparations, reflecting the enhanced need for immune cells in inflammatory conditions. As the reason for this observation we identified a strongly decreased adhesion of BMM to plastic induced by CIA in the acute stage of disease. This effect was accompanied by a strong reduction of adhesion to collagen type I and fibronectin in the chronic stage which may inhibit the in vivo migration of BMM and therefore prevent the induction of new inflammatory processes. NA inhibited adhesion to collagen type I and laminin in BMM from control animals whereas CIA BMM were anergic to NA stimulation. Translation into in vivo conditions suggests that local NA cannot prevent CIA BMM from migration and thus promotes inflammation. Oppositely, NA enhanced plastic adhesion of control BMM and had again no effect on CIA BMM. In CIA BMM, VIP instead leads to enhanced plastic adhesion possibly representing a compensatory mechanism. The results point to CIA as a modulator of NA-driven adhesion to plastic and other molecules of the extracellular matrix, like collagen type I and laminin. Furthermore, starting early after immunization, CIA initiates mechanisms that lead to impaired proliferative activity exclusively at non- and low-inflammatory stages. Inflammatory conditions in vivo may override the underlying effects and restore or even enhance proliferation. Anti-proliferative effects of NA, ACh and VIP on BMM from control rats were altered and dampened by CIA. Regarding caspase 3/7 activity, CIA induced a switch in BMM reactivity towards stimulation with ACh, NA and VIP, clearly protecting BMM from apoptosis induced by these neurotransmitters. These data suggest that CIA stage-dependently induces changes in neurotransmitter reactivity of BMM leading to alterations in metabolic properties like adhesion, apoptosis and proliferation. We also observed changes in osteoclastogenesis of BMM induced by CIA acting rather on activity than on differentiation. We detected inhibition of cathepsin K activity in the cell culture supernatant of osteoclasts generated from BMM of arthritic rats thereby possibly providing a countermeasure of the organism to prevent further bone destruction. Another regulatory mechanism which could prevent excessive osteoclast formation is the enhanced sensitivity of CIA osteoclasts to deprivation of M-CSF and RankL that was apparent early after induction of CIA. Although CIA clearly affects gene expression of osteoclast activity and differentiation markers in a time-dependent manner, those alterations do not become evident on cellular level. Additionally, we suggest that NA and VIP have mostly protective effects preventing bone matrix degradation by inhibition of cathepsin K activity, an observation which is probably attributed to an increased cAMP signaling via Gs protein-coupled receptors. Oppositely, we report an enhancing effect of ACh on cathepsin K and resorptive activity presumably mediated via muscarinic receptors. Increased osteoclast numbers upon ACh stimulation and a dose-dependent decrease in osteoclast numbers and increased caspase 3/7 reactivity by NA/VIP stimulation corroborates the assumption of opposite effects of the cholinergic and adrenergic nervous system on in vitro osteoclastogenesis and consequently CIA severity. We suggest that CIA modulates sensitivity towards neurotransmitter stimulation in osteoclasts enabling adaptation to locally changing neurotransmitter concentrations during disease pathogenesis.

Einleitung: Neben der chirurgischen Therapie stellt die Bestrahlung als primäre sowie adjuvante Therapieoption eine wichtige Säule in der Behandlung von Plattenepithelkarzinomen der Kopf-Hals-Region dar. Unter physiologischen Bedingungen wirken konstitutiv exprimierte Hitzeschockproteine als[zum vollständigen Text gelangen Sie über die oben angegebene URL], 87. Jahresversammlung der Deutschen Gesellschaft für Hals-Nasen-Ohren-Heilkunde, Kopf- und Hals-Chirurgie

Excessive synovial osteoclastogenesis is a hallmark of rheumatoid arthritis (RA). Concomitantly, local synovial changes comprise neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze if collagen-induced arthritis (CIA) alters bone marrow-derived macrophage (BMM) osteoclastogenesis and osteoclast activity, and how sympathetic neurotransmitters participate in this process. Therefore, BMMs from Dark Agouti rats at different CIA stages were differentiated into osteoclasts in vitro and osteoclast number, cathepsin K activity, matrix resorption and apoptosis were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA) vasoactive intestinal peptide (VIP) and assay-dependent, adenylyl cyclase activator NKH477. We observed modulation of neurotransmitter receptor mRNA expression in CIA osteoclasts without affecting protein level. CIA stage-dependently altered marker gene expression associated with osteoclast differentiation and activity without affecting osteoclast number or activity. Neurotransmitter stimulation modulated osteoclast differentiation, apoptosis and activity. VIP, NA and adenylyl cyclase activator NKH477 inhibited cathepsin K activity and osteoclastogenesis (NKH477, 10(-6) M NA) whereas ACh mostly acted pro-osteoclastogenic. We conclude that CIA alone does not affect metabolism of in vitro generated osteoclasts whereas stimulation with NA, VIP plus specific activation of adenylyl cyclase induced anti-resorptive effects probably mediated via cAMP signaling. Contrary, we suggest pro-osteoclastogenic and pro-resorptive properties of ACh mediated via muscarinic receptors.