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.

Adrenoceptor Expression during Intervertebral Disc Degeneration.

Healthy and degenerating intervertebral discs (IVDs) are innervated by sympathetic nerves, however, adrenoceptor (AR) expression and functionality have never been investigated systematically. Therefore, AR gene expression was analyzed in both tissue and isolated cells from degenerated human IVDs. Furthermore, human IVD samples and spine sections of wildtype mice (WT) and of a mouse line that develops spontaneous IVD degeneration (IVDD, in SM/J mice) were stained for ARs and extracellular matrix (ECM) components. In IVD homogenates and cells α1a-, α1b-, α2a-, α2b-, α2c-, ß1-, and ß2-AR genes were expressed. In human sections, ß2-AR was detectable, and its localization parallels with ECM alterations. Similarly, in IVDs of WT mice, only ß2-AR was expressed, and in IVDs of SM/J mice, ß2AR expression was stronger accompanied by increased collagen II, collagen XII, decorin as well as decreased cartilage oligomeric matrix protein expression. In addition, norepinephrine stimulation of isolated human IVD cells induced intracellular signaling via ERK1/2 and PKA. For the first time, the existence and functionality of ARs were demonstrated in IVD tissue samples, suggesting that the sympathicus might play a role in IVDD. Further studies will address relevant cellular mechanisms and thereby help to develop novel therapeutic options for IVDD.

Role of Norepinephrine in IL-1ß-Induced Chondrocyte Dedifferentiation under Physioxia.

As part of the pathogenesis of osteoarthritis (OA), chondrocytes lose their phenotype and become hypertrophic, or dedifferentiate, mainly driven by interleukin-1ß (IL-1ß). The contribution of other factors to the dedifferentiation process is not completely understood. Recent studies suggested a dose-dependent role for the sympathetic neurotransmitter norepinephrine (NE) in OA chondrocyte metabolism. Therefore, the aim of this study was to analyze the contribution of NE (10-8 M, 10-6 M) to human articular OA chondrocyte dedifferentiation in the absence or presence of IL-1ß (0.5 ng/mL). Here, we demonstrate that OA chondrocytes express α2A-, α2C- and ß2-adrenoceptors (AR) and show the characteristic shift towards a fibroblast-like shape at day 7 in physioxic monolayer culture. NE alone did not affect morphology but, in combination with IL-1ß, markedly accelerated this shift. Moderate glycosaminoglycan (GAG) staining was observed in untreated and NE-treated cells, while IL-1ß strongly decreased GAG deposition. IL-1ß alone or in combination with NE decreased SOX9, type II collagen, COMP, and aggrecan, and induced MMP13 and ADAMTS4 gene expression, indicating an accelerated dedifferentiation. NE alone did not influence gene expression and did not modulate IL-1ß-mediated effects. In conclusion, these results indicate that low-grade inflammation exerts a dominant effect on chondrocyte dedifferentiation and should be targeted early in OA therapy.

Norepinephrine Inhibits Synovial Adipose Stem Cell Chondrogenesis via α2a-Adrenoceptor-Mediated ERK1/2 Activation.

In recent years, first evidences emerged that sympathetic neurotransmitters influence osteoarthritis (OA) manifestation. Joint-resident stem cells might contribute to cartilage repair, however, their chondrogenic function is reduced. The neurotransmitter norepinephrine (NE) was detected in the synovial fluid of trauma and OA patients. Therefore, the aim of this study was to analyse how NE influences the chondrogenesis of synovial adipose tissue-derived stem cells (sASCs). sASCs were isolated from knee-OA patients synovia. After adrenoceptor (AR) expression analysis, proliferation and chondrogenic differentiation in presence of NE and/or α- and ß-AR antagonist were investigated. Cell count, viability, chondrogenic and hypertophic gene expression, sulfated glycosaminoglycan (sGAG) and type II collagen content were determined. Key AR-dependent signaling (ERK1/2, PKA) was analyzed via western blot. sASC expressed α1A-, α1B-, α2A-, α2B-, α2C-, and ß2-AR in monolayer and pellet culture. NE did not affect proliferation and viability, but 10-7 and 10-6 M NE significantly reduced sGAG and type II collagen content as well as ERK1/2 phosphorylation. These effects were fully reversed by yohimbine (α2-AR antagonist). Our study confirms the important role of NE in sASC chondrogenic function and provides new insights in OA pathophysiology. Future studies might help to develop novel therapeutic options targeting neuroendocrine pathways for OA treatment.

ß2-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 ß2-AR subtype seems to play a major role during OA progression. However, the importance of ß2-AR has not yet been investigated in knee OA. Therefore, we examined the development of knee OA in ß2-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 ß2-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 ß2-AR deficiency but to a synergistic effect of OA and elevated leptin concentrations. Taken together, ß2-AR plays a major role in OA-related subchondral bone remodeling and is thus an attractive target for the exploration of novel therapeutic avenues.

Platelet-rich fibrin-based matrices to improve angiogenesis in an in vitro co-culture model for bone tissue engineering.

In the context of prevascularization strategies for tissue-engineering purposes, co-culture systems consisting of outgrowth endothelial cells (OECs) and primary osteoblasts (pOBs) have been established as a promising in vitro tool to study regeneration mechanisms and to identify factors that might positively influence repair processes such as wound healing or angiogenesis. The development of autologous injectable platelet-rich fibrin (PRF), which can be generated from peripheral blood in a minimal invasive procedure, fulfils several requirements for clinically applicable cell-based tissue-engineering strategies. During this study, the established co-culture system of OECs and pOBs was mixed with injectable PRF and was cultivated in vitro for 24 h or 7 days. The aim of this study was to analyse whether PRF might have a positive effect on wound healing processes and angiogenic activation of OECs in the co-culture with regard to proinflammatory factors, adhesion molecules and proangiogenic growth factor expression. Histological cell detection revealed the formation of lumina and microvessel-like structures in the PRF/co-culture complexes after 7 days of complex cultivation. Interestingly, the angiogenic activation of OECs was accompanied by an upregulation of wound healing-associated factors, as well as by a higher expression of the proangiogenic factor vascular endothelial growth factor, which was evaluated both on the mRNA level as well as on the protein level. Thus, PRF might positively influence wound healing processes, in particular angiogenesis, in the in vitro co-culture, making autologous PRF-based matrices a beneficial therapeutic tool for tissue-engineering purposes by simply profiting from the PRF, which contains blood plasma, platelets and leukocytes.