The effect of CaV1.2 inhibitor nifedipine on chondrogenic differentiation of human bone marrow or menstrual blood-derived mesenchymal stem cells and chondrocytes

Abstract Cartilage is an avascular tissue and sensitive to mechanical trauma and/or age-related degenerative processes leading to the development of osteoarthritis (OA). Therefore, it is important to investigate the mesenchymal cell-based chondrogenic regenerating mechanisms and possible their regulation. The aim of this study was to investigate the role of intracellular calcium (iCa²⁺) and its regulation through voltage-operated calcium channels (VOCC) on chondrogenic differentiation of mesenchymal stem/stromal cells derived from human bone marrow (BMMSCs) and menstrual blood (MenSCs) in comparison to OA chondrocytes. The level of iCa²⁺ was highest in chondrocytes, whereas iCa²⁺ store capacity was biggest in MenSCs and they proliferated better as compared to other cells. The level of CaV1.2 channels was also highest in OA chondrocytes than in other cells. CaV1.2 antagonist nifedipine slightly suppressed iCa²⁺, Cav1.2 and the proliferation of all cells and affected iCa²⁺ stores, particularly in BMMSCs. The expression of the CaV1.2 gene during 21 days of chondrogenic differentiation was highest in MenSCs, showing the weakest chondrogenic differentiation, which was stimulated by the nifedipine. The best chondrogenic differentiation potential showed BMMSCs (SOX9 and COL2A1 expression); however, purposeful iCa²⁺ and VOCC regulation by blockers can stimulate a chondrogenic response at least in MenSCs

MicroRNAs interfere with DNA methylation in rheumatoid arthritis synovial fibroblasts

BACKGROUND The DNA of rheumatoid arthritis synovial fibroblasts (RASF) is globally hypomethylated; this contributes to an aggressive behaviour. In an attempt to remethylate these cells, we supplemented with methyl donors. We investigated the possible interference of microRNAs (miRs). MATERIAL AND METHODS RASF were treated with L-methionine or betaine. Transcripts of de novo methyltransferases (DNMTs) and miRs were measured by real-time PCR, and a transcription PCR array was performed. Levels of homocysteine, matrix metalloproteinase-1 (MMP-1) and global DNA methylation were determined. Transfection with lipofectamine was performed with specific pre-miRs and anti-miRs, such as miR29 and let7f. RESULTS L-methionine was more efficient to increase DNA methylation than betaine. This was associated with a reduced expression of DNMT3A mRNA in betaine-treated RASF. Betaine increases the expression of miR29 in RASF which targets DNMT3A, thereby limiting the remethylation process. Nevertheless, betaine inhibited the expression of multiple transcription factors, decreased the release of MMP-1, biosynthesis of homocysteine and cell migration. CONCLUSION Alterations in cellular miRs profiles, in particular the upregulation of miR29, which targets DNMT3A, may limit the efficiency of betaine if it is used as DNA remethylating agent. However, L-methionine also has similar impact on miR29 expression. On the other hand, betaine has multiple other beneficial effects on the activated phenotype of RASF; it is not excluded that the effect of betaine on DNMT3A is, at least in part, indirect. Clinical trials with betaine could be promising

The antihypertensive drug nifedipine modulates the metabolism of chondrocytes and human bone marrow-derived mesenchymal stem cells

Abstract Aging is associated with the development of various chronic diseases, in which both cardiovascular disorders and osteoarthritis are dominant. Currently, there is no effective treatment for osteoarthritis, whereas hypertension is often treated with L-type voltage-operated calcium channel blocking drugs, nifedipine being among the most classical ones. Although nifedipine together with other L-type voltage-operated calcium channel inhibitors plays an important role in controlling hypertension, there are unresolved questions concerning its possible effect on cartilage tissue homeostasis and the development of osteoarthritis. The aim of this study was to analyse the effects of nifedipine on metabolic processes in human chondrocytes and bone marrow mesenchymal stem cells. To better understand whether the metabolic effects are mediated specifically through L-type voltage-operated calcium channel, effects of the agonist BayK8644 were analyzed in parallel. Nifedipine downregulated and mitochondrial respiration and ATP production in both cell types. Analysis of cartilage explants by electron microscopy also suggested that a small number of chondrocyte mitochondria's lose their activity in response to nifedipine. Conversely, nifedipine enhanced glycolytic capacity in chondrocytes, suggesting that these cells have the capacity to switch from oxidative phosphorylation to glycolysis and alter their metabolic activity in response to L-type voltage-operated calcium channel inhibition. Such a metabolic switch was not observed in bone marrow mesenchymal stem cells. Nitric oxide activity was upregulated by nifedipine in bone marrow mesenchymal stem cells and particularly in chondrocytes, implying its involvement in the effects of nifedipine on metabolism in both tested cell types. Furthermore, stimulation with nifedipine resulted in elevated production of collagen type II and glycosaminoglycans in micromass cultures under chondrogenic conditions. Taken together, we conclude that the antihypertensive drug nifedipine inhibits mitochondrial respiration in both chondrocytes and bone marrow mesenchymal stem cells and that these effects may be associated with the increased nitric oxide accumulation and pro-inflammatory activity. Nifedipine had positive effects on the production of collagen type II and proteoglycans in both cell types, implying potentially beneficial anabolic responses in articular cartilage. These results highlight a potential link between antihypertensive drugs and cartilage health

Different phenotypes and chondrogenic responses of human menstrual blood and bone marrow mesenchymal stem cells to activin A and TGF-β3

Abstract Background: Due to its low capacity for self-repair, articular cartilage is highly susceptible to damage and deterioration, which leads to the development of degenerative joint diseases such as osteoarthritis (OA). Menstrual blood-derived mesenchymal stem/stromal cells (MenSCs) are much less characterized, as compared to bone marrow mesenchymal stem/stromal cells (BMMSCs). However, MenSCs seem an attractive alternative to classical BMMSCs due to ease of access and broader differentiation capacity. The aim of this study was to evaluate chondrogenic differentiation potential of MenSCs and BMMSCs stimulated with transforming growth factor β (TGF-β3) and activin A. Methods: MenSCs (n = 6) and BMMSCs (n = 5) were isolated from different healthy donors. Expression of cell surface markers CD90, CD73, CD105, CD44, CD45, CD14, CD36, CD55, CD54, CD63, CD106, CD34, CD10, and Notch1 was analyzed by flow cytometry. Cell proliferation capacity was determined using CCK-8 proliferation kit and cell migration ability was evaluated by scratch assay. Adipogenic differentiation capacity was evaluated according to Oil-Red staining and osteogenic differentiation according to Alizarin Red staining. Chondrogenic differentiation (activin A and TGF-β3 stimulation) was investigated in vitro and in vivo (subcutaneous scaffolds in nude BALB/c mice) by expression of chondrogenic genes (collagen type II, aggrecan), GAG assay and histologically. Activin A protein production was evaluated by ELISA during chondrogenic differentiation in monolayer culture. Results: MenSCs exhibited a higher proliferation rate, as compared to BMMSCs, and a different expression profile of several cell surface markers. Activin A stimulated collagen type II gene expression and glycosaminoglycan synthesis in TGF-β3 treated MenSCs but not in BMMSCs, both in vitro and in vivo, although the effects of TGF-β3 alone were more pronounced in BMMSCs in vitro. Conclusion: These data suggest that activin A exerts differential effects on the induction of chondrogenic differentiation in MenSCs vs. BMMSCs, which implies that different mechanisms of chondrogenic regulation are activated in these cells. Following further optimization of differentiation protocols and the choice of growth factors, potentially including activin A, MenSCs may turn out to be a promising population of stem cells for the development of cell-based therapies with the capacity to stimulate cartilage repair and regeneration in OA and related osteoarticular disorders

Emerging technologies and platforms for the immunodetection of multiple biochemical markers in osteoarthritis research and therapy

Abstract Biomarkers, especially biochemical markers, are important in osteoarthritis (OA) research, clinical trials, and drug development and have potential for more extensive use in therapeutic monitoring. However, they have not yet had any significant impact on disease diagnosis and follow-up in a clinical context. Nevertheless, the development of immunoassays for the detection and measurement of biochemical markers in OA research and therapy is an active area of research and development. The evaluation of biochemical markers representing low-grade inflammation or extracellular matrix turnover may permit OA prognosis and expedite the development of personalized treatment tailored to fit particular disease severities. However, currently detection methods have failed to overcome specific hurdles such as low biochemical marker concentrations, patient-specific variation, and limited utility of single biochemical markers for definitive characterization of disease status. These challenges require new and innovative approaches for development of detection and quantification systems that incorporate clinically relevant biochemical marker panels. Emerging platforms and technologies that are already on the way to implementation in routine diagnostics and monitoring of other diseases could potentially serve as good technological and strategic examples for better assessment of OA. State-of-the-art technologies such as advanced multiplex assays, enhanced immunoassays, and biosensors ensure simultaneous screening of a range of biochemical marker targets, the expansion of detection limits, low costs, and rapid analysis. This paper explores the implementation of such technologies in OA research and therapy. Application of novel immunoassay-based technologies may shed light on poorly understood mechanisms in disease pathogenesis and lead to the development of clinically relevant biochemical marker panels. More sensitive and specific biochemical marker immunodetection will complement imaging biomarkers and ensure evidence-based comparisons of intervention efficacy. We discuss the challenges hindering the development, testing, and implementation of new OA biochemical marker assays utilizing emerging multiplexing technologies and biosensors