Semaphorin 3A-Neuropilin-1 Signaling Modulates MMP13 Expression in Human Osteoarthritic Chondrocytes.

Osteoarthritis (OA) is a complex disorder of diarthrodial joints caused by multiple risk factors and is characterized by articular cartilage destruction as well as changes in other articular tissues. Semaphorin 3A (Sema3A), known to be a chemo-repellent for sensory nerve fibers, has recently been implicated in cartilage OA pathophysiology. We demonstrated that the expression of SEMA3A and its receptor neuropilin-1 (NRP1) are synchronously upregulated in chondrocytes isolated from knee cartilage of OA patients compared to non-OA control chondrocytes. In addition, we observed that during in vitro passaging of OA chondrocytes, the Nrp-1 level increases, whereas the Sema3A level decreases. In this study, we aimed to uncover how Sema3A-Nrp-1 signaling affects metabolism and viability of OA chondrocytes via siRNA-mediated inhibition of Nrp-1 expression. We observed a decreased proliferation rate and an increase in adhesion and senescence after Nrp-1 silencing. Moreover, MMP13 gene expression was reduced by approximately 75% in NRP1 knockdown OA chondrocytes, whereas MMP13 expression was induced by Sema3A treatment in control (nt siRNA) OA chondrocytes, accompanied by an impaired AKT phosphorylation. These findings suggest a potential catabolic function of Sema3A signaling in OA chondrocytes by inducing MMP13 expression and by compromising pro-survival AKT activation. We propose that targeting the Sema3A-Nrp-1 signaling axis might be an opportunity to interfere with OA pathogenesis and progression.

Influence of the Peripheral Nervous System on Murine Osteoporotic Fracture Healing and Fracture-Induced Hyperalgesia.

Osteoporotic fractures are often linked to persisting chronic pain and poor healing outcomes. Substance P (SP), α-calcitonin gene-related peptide (α-CGRP) and sympathetic neurotransmitters are involved in bone remodeling after trauma and nociceptive processes, e.g., fracture-induced hyperalgesia. We aimed to link sensory and sympathetic signaling to fracture healing and fracture-induced hyperalgesia under osteoporotic conditions. Externally stabilized femoral fractures were set 28 days after OVX in wild type (WT), α-CGRP- deficient (α-CGRP -/-), SP-deficient (Tac1-/-) and sympathectomized (SYX) mice. Functional MRI (fMRI) was performed two days before and five and 21 days post fracture, followed by µCT and biomechanical tests. Sympathectomy affected structural bone properties in the fracture callus whereas loss of sensory neurotransmitters affected trabecular structures in contralateral, non-fractured bones. Biomechanical properties were mostly similar in all groups. Both nociceptive and resting-state (RS) fMRI revealed significant baseline differences in functional connectivity (FC) between WT and neurotransmitter-deficient mice. The fracture-induced hyperalgesia modulated central nociception and had robust impact on RS FC in all groups. The changes demonstrated in RS FC in fMRI might potentially be used as a bone traumata-induced biomarker regarding fracture healing under pathophysiological musculoskeletal conditions. The findings are of clinical importance and relevance as they advance our understanding of pain during osteoporotic fracture healing and provide a potential imaging biomarker for fracture-related hyperalgesia and its temporal development. Overall, this may help to reduce the development of chronic pain after fracture thereby improving the treatment of osteoporotic fractures.

Induction of ALP and MMP9 activity facilitates invasive behavior in heterogeneous human BMSC and HNSCC 3D spheroids.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells capable of differentiating into adipocytic, osteogenic, chondrogenic, and myogenic lineages. There is growing evidence that MSCs home into the tumor microenvironment attracted by a variety of signals such as chemokines, growth factors, and cytokines. Tumor-homing stem cells may originate from bone marrow-derived MSCs (BMSCs) or adipose tissue-derived MSCs. Recent scientific data suggest that MSCs in combination with tumor cells can either promote or inhibit tumorigenic behavior. In head and neck squamous cell carcinoma (HNSCC), BMSCs are reported to be enriched with a potential negative role. Here, we evaluated the effect of BMSCs from 4 different donors in combination with 4 HNSCC cell lines in a 3-dimensional multicellular spheroid model. Heterogeneous combinations revealed an up-regulation of gene and protein expression of osteogenic markers runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP) together with a substantial secretion of matrix metalloproteinase 9. Moreover, heterogenous BMSC/tumor spheroids showed increased invasion compared with homogenous spheroids in a Boyden chamber invasion assay. Furthermore, inhibition of ALP resulted in a substantially decreased spreading of heterogeneous spheroids on laminin-rich matrix. In summary, our data suggest a prometastatic effect of BMSCs combined with HNSCC.-Wessely, A., Waltera, A., Reichert, T. E., Stöckl, S., Grässel, S., Bauer, R. J. Induction of ALP and MMP9 activity facilitates invasive behavior in heterogeneous human BMSC and HNSCC 3D-spheroids.

Early OA Stage Like Response Occurs after Dynamic Stretching of Human Synovial Fibroblasts.

As events triggering early osteoarthritis onset can be related to mechanical stress and proinflammatory signaling, we investigated the effect of different mechanical strain protocols on the expression of proinflammatory genes, as well as extracellular matrix remodelling in human synovial fibroblasts. Three distinct models of tensile stretching were applied: static isotropic tensile strain at 0 Hz, 16% tension for 48 h; short-term high-frequency cyclic tension at 1 Hz, 10% tension for 4 h; and dynamic tensile stretching for 48 h, consisting of two blocks of moderate stretching at 0.2 Hz, 2%, advanced stretching at 0.5 Hz, 15%, or a combination of both. General signs of inflammation were present after static isotropic tension, whereas short-term high-frequency cyclic tension showed increased levels of IL-6 paired with diminished levels of IL-1ß. Reduced inflammatory effects of TNF-α, IL-6, and IL-1ß were observed when exposed to advanced stretching. Long-term tensile strain induced extracellular matrix remodelling at the gene and protein levels. While hyaluronan acid synthesis was increased with static tensile strain, dynamic tensile stretching had a reducing effect. Our study revealed that proinflammatory markers were activated by mechanical strain as seen in static isotropic tension and short-term high-frequency tensile strain, whereas long-term exposure induced extracellular matrix remodelling processes.

Impact of the Sensory and Sympathetic Nervous System on Fracture Healing in Ovariectomized Mice.

The peripheral nervous system modulates bone repair under physiological and pathophysiological conditions. Previously, we reported an essential role for sensory neuropeptide substance P (SP) and sympathetic nerve fibers (SNF) for proper fracture healing and bone structure in a murine tibial fracture model. A similar distortion of bone microarchitecture has been described for mice lacking the sensory neuropeptide α-calcitonin gene-related peptide (α-CGRP). Here, we hypothesize that loss of SP, α-CGRP, and SNF modulates inflammatory and pain-related processes and also affects bone regeneration during fracture healing under postmenopausal conditions. Intramedullary fixed femoral fractures were set to 28 days after bilateral ovariectomy (OVX) in female wild type (WT), SP-, α-CGRP-deficient, and sympathectomized (SYX) mice. Locomotion, paw withdrawal threshold, fracture callus maturation and numbers of TRAP-, CD4-, CD8-, F4/80-, iNos-, and Arg1-positive cells within the callus were analyzed. Nightly locomotion was reduced in unfractured SP-deficient and SYX mice after fracture. Resistance to pressure was increased for the fractured leg in SP-deficient mice during the later stages of fracture healing, but was decreased in α-CGRP-deficient mice. Hypertrophic cartilage area was increased nine days after fracture in SP-deficient mice. Bony callus maturation was delayed in SYX mice during the later healing stages. In addition, the number of CD 4-positive cells was reduced after five days and the number of CD 8-positive cells was additionally reduced after 21 days in SYX mice. The number of Arg1-positive M2 macrophages was higher in α-CGRP-deficient mice five days after fracture. The alkaline phosphatase level was increased in SYX mice 16 days after fracture. Absence of α-CGRP appears to promote M2 macrophage polarization and reduces the pain threshold, but has no effect on callus tissue maturation. Absence of SP reduces locomotion, increases the pain-threshold, and accelerates hypertrophic callus tissue remodeling. Destruction of SNF reduces locomotion after fracture and influences bony callus tissue remodeling during the later stages of fracture repair, whereas pain-related processes are not affected.

Norepinephrine Inhibits the Proliferation of Human Bone Marrow-Derived Mesenchymal Stem Cells via ß2-Adrenoceptor-Mediated ERK1/2 and PKA Phosphorylation.

Bone marrow-derived mesenchymal stem cells (BMSCs) represent an alternative to chondrocytes to support cartilage regeneration in osteoarthritis (OA). The sympathetic neurotransmitter norepinephrine (NE) has been shown to inhibit their chondrogenic potential; however, their proliferation capacity under NE influence has not been studied yet. Therefore, we used BMSCs obtained from trauma and OA donors and compared the expression of adrenergic receptors (AR). Then, BMSCs from both donor groups were treated with NE, as well as with combinations of NE and α1-, α2- or ß1/2-AR antagonists (doxazosin, yohimbine or propranolol). Activation of AR-coupled signaling was investigated by analyzing ERK1/2 and protein kinase A (PKA) phosphorylation. A similar but not identical subset of ARs was expressed in trauma (α2B-, α2C- and ß2-AR) and OA BMSCs (α2A-, α2B-, and ß2-AR). NE in high concentrations inhibited the proliferation of both trauma and OA BMCSs significantly. NE in low concentrations did not influence proliferation. ERK1/2 as well as PKA were activated after NE treatment in both BMSC types. These effects were abolished only by propranolol. Our results demonstrate that NE inhibits the proliferation and accordingly lowers the regenerative capacity of human BMSCs likely via ß2-AR-mediated ERK1/2 and PKA phosphorylation. Therefore, targeting ß2-AR-signaling might provide novel OA therapeutic options.

SOX9 Knockout Induces Polyploidy and Changes Sensitivity to Tumor Treatment Strategies in a Chondrosarcoma Cell Line.

As most chemotherapeutic drugs are ineffective in the treatment of chondrosarcoma, we studied the expression pattern and function of SOX9, the master transcription factor for chondrogenesis, in chondrosarcoma, to understand the basic molecular principles needed for engineering new targeted therapies. Our study shows an increase in SOX9 expression in chondrosarcoma compared to normal cartilage, but a decrease when the tumors are finally defined as dedifferentiated chondrosarcoma (DDCS). In DDCS, SOX9 is almost completely absent in the non-chondroid, dedifferentiated compartments. CRISPR/Cas9-mediated knockout of SOX9 in a human chondrosarcoma cell line (HTB94) results in reduced proliferation, clonogenicity and migration, accompanied by an inability to activate MMP13. In contrast, adhesion, apoptosis and polyploidy formation are favored after SOX9 deletion, probably involving BCL2 and survivin. The siRNA-mediated SOX9 knockdown partially confirmed these results, suggesting the need for a certain SOX9 threshold for particular cancer-related events. To increase the efficacy of chondrosarcoma therapies, potential therapeutic approaches were analyzed in SOX9 knockout cells. Here, we found an increased impact of doxorubicin, but a reduced sensitivity for oncolytic virus treatment. Our observations present novel insight into the role of SOX9 in chondrosarcoma biology and could thereby help to overcome the obstacle of drug resistance and limited therapy options.

Osteoarthritis and Cartilage Regeneration: Focus on Pathophysiology and Molecular Mechanisms.

Osteoarthritis (OA) is a leading cause of disability and source of societal cost in older adults [...].

Impact of Mechanical Load on the Expression Profile of Synovial Fibroblasts from Patients with and without Osteoarthritis.

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 (TNFα, 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 TNFα 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.

Sensory Neuropeptides and their Receptors Participate in Mechano-Regulation of Murine Macrophages.

This study aimed to analyze if the sensory neuropeptide SP (SP) and the neurokinin receptor 1 (NK1R) are involved in macrophage mechano-transduction, similar to chondrocytes, and if alpha-calcitonin gene-related peptide (αCGRP) and the CGRP receptor (CRLR/Ramp1) show comparable activity. Murine RAW264.7 macrophages were subjected to a cyclic stretch for 1⁻3 days and 4 h/day. Loading and neuropeptide effects were analyzed for gene and protein expression of neuropeptides and their receptors, adhesion, apoptosis, proliferation and ROS activity. Murine bone marrow-derived macrophages (BMM) were isolated after surgical osteoarthritis (OA) induction and proliferation, apoptosis and osteoclastogenesis were analyzed in response to loading. Loading induced NK1R and CRLR/Ramp1 gene expression and altered protein expression in RAW264.7 macrophages. SP protein and mRNA level decreased after loading whereas αCGRP mRNA expression was stabilized. SP reduced adhesion in loaded RAW264.7 macrophages and both neuropeptides initially increased the ROS activity followed by a time-dependent suppression. OA induction sensitized BMM to caspase 3/7 mediated apoptosis after loading. Both sensory neuropeptides, SP and αCGRP, and their receptors are involved in murine macrophage mechano-transduction affecting neuropeptide impact on adhesion and ROS activity. OA induction altered BMM apoptosis in response to loading indicate that OA-associated biomechanical alterations might affect the macrophage population.

Do Neuroendocrine Peptides and Their Receptors Qualify as Novel Therapeutic Targets in Osteoarthritis?

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 ß-endorphin (ß-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.

Peripheral Nerve Fibers and Their Neurotransmitters in Osteoarthritis Pathology.

The importance of the nociceptive nervous system for maintaining tissue homeostasis has been known for some time, and it has also been suggested that organogenesis and tissue repair are under neuronal control. Changes in peripheral joint innervation are supposed to be partly responsible for degenerative alterations in joint tissues which contribute to development of osteoarthritis. Various resident cell types of the musculoskeletal system express receptors for sensory and sympathetic neurotransmitters, allowing response to peripheral neuronal stimuli. Among them are mesenchymal stem cells, synovial fibroblasts, bone cells and chondrocytes of different origin, which express distinct subtypes of adrenoceptors (AR), receptors for vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP). Some of these cell types synthesize and secrete neuropeptides such as SP, and they are positive for tyrosine-hydroxylase (TH), the rate limiting enzyme for biosynthesis of catecholamines. Sensory and sympathetic neurotransmitters are involved in the pathology of inflammatory diseases such as rheumatoid arthritis (RA) which manifests mainly in the joints. In addition, they seem to play a role in pathogenesis of priori degenerative joint disorders such as osteoarthritis (OA). Altogether it is evident that sensory and sympathetic neurotransmitters have crucial trophic effects which are critical for joint tissue and bone homeostasis. They modulate articular cartilage, subchondral bone and synovial tissue properties in physiological and pathophysiological conditions, in addition to their classical neurological features.

The NC11 domain of human collagen XVI induces vasculogenic mimicry in oral squamous cell carcinoma cells.

Collagen XVI, a fibril-associated collagen with interrupted triple helix (FACIT) collagen, is involved in oral squamous cell carcinoma (OSCC) and glioblastoma progression. The NC11 domain of collagen XVI has been described previously with a strong implication in physiological processes. We detected the non-collagenous (NC) 11-domain in supernatants of OSCC cells after recombinant expression of full-length collagen XVI and in sera from OSCC patients and healthy individuals. Stable expression of NC11-green fluorescent protein (GFP) fusion protein in OSCC cells initiated proliferation control and block of anchorage-independent growth. Moreover, the NC11 domain triggered the generation of tubular-like net structures on laminin-rich matrix in contrast to mock-GFP control cells and cells expressing full-length collagen XVI. Taqman® quantitative PCR and diaminobenzidine staining in 2D- and 3D cell culture revealed a significantly increased gene and protein expression of VEGFR1, VEGFR2 and uPAR in recombinant NC11-GFP-expressing cells. Specific VEGF receptor inhibition with Axitinib or fetal calf serum heat inactivation prevented formation of tubular-like net structures. Accordantly, NC11-GFP coated culture slides led to an increase of focal adhesion contact formation and the upregulation of VEGFR1 and uPAR in three different non-transfected OSCC cell lines. In summary, we suggest that the NC11 domain of collagen XVI is a potential biomarker for OSCC and triggers vasculogenic mimicry via upregulation of endothelial receptors VEGFR1, VEGFR2 and uPAR in 2D- and 3D OSCC cell culture conditions.

Sox9 modulates cell survival and adipogenic differentiation of multipotent adult rat mesenchymal stem cells.

Sox9 is a key transcription factor in early chondrogenesis with distinct roles in differentiation processes and during embryonic development. Here, we report that Sox9 modulates cell survival and contributes to the commitment of mesenchymal stem cells (MSC) to adipogenic or osteogenic differentiation lineages. We found that the Sox9 activity level affects the expression of the key transcription factor in adipogenic differentiation, C/EBPß, and that cyclin D1 mediates the expression of the osteogenic marker osteocalcin in undifferentiated adult bone-marrow-derived rat MSC. Introducing a stable Sox9 knockdown into undifferentiated rat MSC resulted in a marked decrease in proliferation rate and an increase in apoptotic activity. This was linked to a profound upregulation of p21 and cyclin D1 gene and protein expression accompanied by an induction of caspase 3/7 activity and an inhibition of Bcl-2. We observed that Sox9 silencing provoked a delayed S-phase progression and an increased nuclear localization of p21. The protein stability of cyclin D1 was induced in the absence of Sox9 presumably as a function of altered p38 signalling. In addition, the major transcription factor for adipogenic differentiation, C/EBPß, was repressed after silencing Sox9. The nearly complete absence of C/EBPß protein as a result of increased destabilization of the C/EBPß mRNA and the impact on osteocalcin gene expression and protein synthesis, suggests that a delicate balance of Sox9 level is not only imperative for proper chondrogenic differentiation of progenitor cells, but also affects the adipogenic and probably osteogenic differentiation pathways of MSC. Our results identified Sox9 as an important link between differentiation, proliferation and apoptosis in undifferentiated adult rat mesenchymal stem cells, emphasizing the importance of the delicate balance of a precisely regulated Sox9 activity in MSC not only for proper skeletal development during embryogenesis but probably also for successful repair and regeneration of tissues and organs in adults.

Catecholaminergic-to-cholinergic transition of sympathetic nerve fibers is stimulated under healthy but not under inflammatory arthritic conditions.

OBJECTIVE: Density of sympathetic nerve fibers decreases in inflamed arthritic tissue tested by immunoreactivity towards tyrosine-hydroxylase (TH, catecholaminergic key enzyme). Since sympathetic nerve fibers may change phenotype from catecholaminergic to cholinergic (example: sweat glands), loss of nerve fibers may relate to undetectable TH. We aimed to investigate possible catecholaminergic-to-cholinergic transition of sympathetic nerve fibers in synovial tissue of animals with arthritis, and patients with rheumatoid arthritis (RA) and osteoarthritis (OA), and we wanted to find a possible transition factor. METHODS: Nerve fibers were detected by immunofluorescence towards TH (catecholaminergic) and vesicular acetylcholine transporter (cholinergic). Co-culture experiments with sympathetic ganglia and lymphocytes or osteoclast progenitors were designed to find stimulators of catecholaminergic-to-cholinergic transition (including gene expression profiling). RESULTS: In mouse joints, an increased density of cholinergic relative to catecholaminergic nerve fibers appeared towards day 35 after immunization, but most nerve fibers were located in healthy joint-adjacent skin or muscle and almost none in inflamed synovial tissue. In humans, cholinergic fibers are more prevalent in OA synovial tissue than in RA. Co-culture of sympathetic ganglia with osteoclast progenitors obtained from healthy but not from arthritic animals induced catecholaminergic-to-cholinergic transition. Osteoclast mRNA microarray data indicated that leukemia inhibitory factor (LIF) is a candidate transition factor, which was confirmed in ganglia experiments, particularly, in the presence of progesterone. CONCLUSION: In humans and mice, catecholaminergic-to-cholinergic sympathetic transition happens in less inflamed tissue but not in inflamed arthritic tissue. Under healthy conditions, presence of cholinergic sympathetic nerve fibers may support the cholinergic anti-inflammatory influence recently described.

The impact of hypoxia on mesenchymal progenitor cells of human skeletal tissue in the pathogenesis of heterotopic ossification.

PURPOSE: Mesenchymal progenitor cells (MPCs) are capable of differentiating into osteo/chondrogenic cells to contribute substantially to heterotopic ossification (HO). This study aimed to examine the impact of hypoxia on MPCs in the aetiology of HO. METHODS: MPCs from human normal and HO skeletal tissue were cultivated under normoxia and hypoxia. Gene expression of factors which have a key role in HO aetiology (BMPs, COX-1 and COX-2, etc.) were examined by real-time PCR. Tissue of both groups was analysed by immunohistochemistry. RESULTS: Under hypoxia, COX-1, -2 and SOX-9 gene expression was elevated in HO MPCs, whereas in normal muscle tissue only COX-2 was upregulated. MPCs from HO had a significantly elevated gene expression of BMP-4 and decreased expression of BMP-1 and HIF-1 under hypoxia compared to normal MPCs. Immunohistochemistry detected no significant differences between normal and HO tissue. CONCLUSIONS: Hypoxia causes an enhanced gene expression of factors, which have a key role in HO pathophysiology. A better understanding of this entity will possibly allow reducing HO rates in orthopaedic and trauma surgery.

IL-7 receptor α expressing B cells act proinflammatory in collagen-induced arthritis and are inhibited by sympathetic neurotransmitters.

OBJECTIVES: The sympathetic nervous system (SNS) as well as the interleukin (IL)-7/IL-7 receptor (IL-7R) system play a role in the pathogenesis of arthritis. However, the target cells and mechanisms involved are not fully resolved. The goal of this study was to determine if B cells are influenced by IL-7 and to investigate the possible interplay between the SNS and the IL-7/IL-7R system on B cells in arthritis. METHODS: Collagen type II-induced arthritis (CIA) in DBA1 mice. ELISA to determine specific anti-CII antibodies. Fluorescence activated cell sorting (FACS) analysis to determine IL-7R+ cells and intracellular phosphorylated signal transducer and activator of transcription 5 (pSTAT5). Immunohistochemistry to show IL-7R+ B cells in rheumatoid arthritis (RA) and osteoarthritis (OA) synovial tissue. RESULTS: IL-7 stimulated IL-7R+ mature B cells act proinflammatory (increased clinical score, increased anticollagen type II antibodies) after cell transfer in CIA. The sympathetic neurotransmitter norepinephrine abrogates this effect. Expression of IL-7Rα is increased when B cells are activated (anti-CD40 or lipopolysaccharide) in vitro and stimulating the IL-7R induces intracellular accumulation of pSTAT5. α- And ß-adrenergic agonists show no influence on expression levels of IL-7R on activated B cells; however, intracellular IL-7R downstream signalling is abrogated via the ß2-adreonceptor (ß2AR) agonist terbutaline. IL-7R and ß2AR are also expressed on B cells in synovial tissue from RA and OA patients. CONCLUSIONS: These data indicate that IL7R+ B cells have a proinflammatory role in arthritis which can be inhibited by the sympathetic neurotransmitter norepinephrine via inhibition of IL-7R signalling.

Tissue-engineering strategies to repair chondral and osteochondral tissue in osteoarthritis: use of mesenchymal stem cells.

Focal chondral or osteochondral lesions can be painful and disabling because they have insufficient intrinsic repair potential, and constitute one of the major extrinsic risk factors for osteoarthritis (OA). Attention has, therefore, been paid to regenerative therapeutic procedures for the early treatment of cartilaginous defects. Current treatments for OA are not regenerative and have little effect on the progressive degeneration of joint tissue. One major reason for this underrepresentation of regenerative therapy is that approaches to treating OA with cell-based strategies have to take into consideration the larger sizes of the defects, as compared with isolated focal articular-cartilage defects, and the underlying disease process. Here, we review current treatment strategies using mesenchymal stem cells (MSCs) for chondral and osteochondral tissue repair in trauma and OA-affected joints. We discuss tissue-engineering approaches, in preclinical large-animal models and clinical studies in humans, which use crude bone-marrow aspirates and MSCs from different tissue sources in combination with bioactive agents and materials.

Inhibition of caspase-11 under inflammatory conditions suppresses chondrogenic differentiation.

Caspase-11 is the murine homologue of human caspases-4 and -5 and is involved in mediating the inflammatory response. However, its functions are often confused and misinterpreted with the more important and better described caspase-1. Therefore, this study focused exclusively on the specific roles of caspase-11, both in cartilage formation and in the inflammatory environment. The presence of caspase-11 during mouse limb development and in chondrogenic cell cultures was investigated by immunofluorescence detection. Subsequently, the function of caspase-11 was downregulated and the affected molecules investigated. The expression analysis applied for osteo/chondrogenesis associated factors and inflammatory cytokines. Simultaneously, morphological appearance of the micromass cultures was evaluated. The results revealed that caspase-11 is physiologically present during cartilage development, but its inhibition under physiological conditions has no significant effect on chondrogenic differentiation. However, in an inflammatory environment, inhibition and downregulation of caspase-11 leads to reduced differentiation of cartilage nodules. Additionally, reduced expression of several genes including Col2a1 and Sp7 and conversely increased expression of Mmp9 were observed. In the cytokine expression panel, a significant decrease was found in molecules that, along with the inflammatory function, may also be involved in cartilage differentiation. The findings bring new information about caspase-11 in chondrogenesis and show that its downregulation under inflammatory conditions reduces cartilage formation.

Sox9 Modulates proliferation and expression of osteogenic markers of adipose-derived stem cells (ASC).

BACKGROUND: Mesenchymal stem cells (MSC) are promising tools for tissue-engineering and musculoskeletal regeneration. They reside within various tissues, like adipose tissue, periosteum, synovia, muscle, dermis, blood and bone marrow, latter being the most common tissue used for MSC isolation. A promising alternative source for MSC is adipose tissue due to better availability and higher yield of MSC in comparison to bone marrow. A drawback is the yet fragmentary knowledge of adipose-derived stem cell (ASC) physiology in order to make them a safe tool for in vivo application. METHODS/RESULTS: Here, we identified Sox9 as a highly expressed and crucial transcription factor in undifferentiated rat ASC (rASC). In comparison to rat bone marrow-derived stem cells (rBMSC), mRNA and protein levels of Sox9 were significantly higher in rASC. To study the role of Sox9 in detail, we silenced Sox9 with shRNA in rASC and examined proliferation, apoptosis and the expression of osteogenic differentiation markers. Our results clearly point to a difference in the expression profile of osteogenic marker genes between undifferentiated rASC and rBMSC in early passages. Sox9 silencing induced the expression of osteocalcin, Vegfα and Mmp13, and decreased rASC proliferation accompanied with an induction of p21 and cyclin D1 expression and delayed S-phase entry. CONCLUSIONS: We suggest a pro-proliferative role for Sox9 in undifferentiated rASC which may explain the higher proliferation rate of rASC compared to rBMSC. Moreover, we propose an osteogenic differentiation delaying role of Sox9 in rASC which suggests that Sox9 expression is needed to maintain rASC in an undifferentiated, proliferative state.