Bone metabolism and inflammatory biomarkers in radiographic and non-radiographic axial spondyloarthritis patients: a comprehensive evaluation

IntroductionAxial spondyloarthritis (axSpA) is a heterogeneous disease that can be represented by radiographic axSpA (r-axSpA) and non-radiographic axSpA (nr-axSpA). This study aimed to evaluate the relationship between the markers of inflammation and bone turnover in r-axSpA patients and nr-axSpA patients.MethodsA cross-sectional study included 29 r-axSpA patients, 10 nr-axSpA patients, and 20 controls matched for age and sex. Plasma markers related to bone remodeling such as human procollagen type 1 N-terminal propeptide (P1NP), sclerostin, tartrate-resistant acid phosphatase 5b (TRACP5b), receptor activator of nuclear factor kappa B ligand (RANKL), and osteoprotegerin (OPG) were measured by an ELISA kit. A panel of 92 inflammatory molecules was analyzed by proximity extension assay.ResultsR-axSpA patients had decreased plasma levels of P1NP, a marker of bone formation, compared to controls. In addition, r-axSpA patients exhibited decreased plasma levels of sclerostin, an anti-anabolic bone hormone, which would not explain the co-existence of decreased plasma P1NP concentration; however, sclerostin levels could also be influenced by inflammatory processes. Plasma markers of osteoclast activity were similar in all groups. Regarding inflammation-related molecules, nr-axSpA patients showed increased levels of serum interleukin 13 (IL13) as compared with both r-axSpA patients and controls, which may participate in the prevention of inflammation. On the other hand, r-axSpA patients had higher levels of pro-inflammatory molecules compared to controls (i.e., IL6, Oncostatin M, and TNF receptor superfamily member 9). Correlation analysis showed that sclerostin was inversely associated with IL6 and Oncostatin M among others.ConclusionAltogether, different inflammatory profiles may play a role in the development of the skeletal features in axSpA patients particularly related to decreased bone formation. The relationship between sclerostin and inflammation and the protective actions of IL13 could be of relevance in the axSpA pathology, which is a topic for further investigation

TGF-β Prevents Phosphate-Induced Osteogenesis through Inhibition of BMP and Wnt/β-Catenin Pathways

Background: Transforming growth factor-b (TGF-b) is a key cytokine during differentiation of mesenchymal stem cells (MSC) into vascular smooth muscle cells (VSMC). High phosphate induces a phenotypic transformation of vascular smooth muscle cells (VSMC) into osteogenic-like cells. This study was aimed to evaluate signaling pathways involved during VSMC differentiation of MSC in presence or not of high phosphate. Results: Our results showed that TGF-b induced nuclear translocation of Smad3 as well as the expression of vascular smooth muscle markers, such as smooth muscle alpha actin, SM22a, myocardin, and smooth muscle-myosin heavy chain. The addition of high phosphate to MSC promoted nuclear translocation of Smad1/5/8 and the activation of canonical Wnt/bcatenin in addition to an increase in BMP-2 expression, calcium deposition and alkaline phosphatase activity. The administration of TGF-b to MSC treated with high phosphate abolished all these effects by inhibiting canonical Wnt, BMP and TGF-b pathways. A similar outcome was observed in high phosphate-treated cells after the inhibition of canonical Wnt signaling with Dkk-1. Conversely, addition of both Wnt/b-catenin activators CHIR98014 and lithium chloride enhanced the effect of high phosphate on BMP-2, calcium deposition and alkaline phosphatase activity. Conclusions: Full VSMC differentiation induced by TGF-b may not be achieved when extracellular phosphate levels are high. Moreover, TGF-b prevents high phosphate-induced osteogenesis by decreasing the nuclear translocation of Smad 1/5/8 and avoiding the activation of Wnt/b-catenin pathway

Procaine impairs osteogenic maturation of mesenchymal stem cells (MSC).

<p>mRNA expression of mature markers of oste/odontoblast such as A) DMP1 and B) RANKL were measured by RT-PCR after 21 days in culture. The expression in mesenchymal stem cell differentiated into osteo/odontoblasts (OB) was significantly higher than in undifferentiated cells (UC). This expression was reduced by the addition of Procaine (OB+Proc) during the differentiation process. Ribosomal 18S expression was used as housekeeping (* p<0.05 vs all groups).</p

Mechanism of action of procaine.

<p>Changes in mRNA expression of GSK3β from undifferentiated mesenchymal stem cells (UC), MSC differentiated into osteo/odontoblast (OB), MSC differentiated into osteo/odontoblast plus procaine (1μM) (OB+Proc), MSC differentiated into osteo/odontoblast plus lithium chloride (10 mM) (OB+LiCl) and the combination of osteo/odontogenic stimuli, Procaine (1μM) and Lithium Chloride (10 mM) were analyzed A). Effect of increasing concentrations of Procaine (0, 0.5, 1 and 2 μM) on GSK3βexpression B) and phospho-β-catenin C) for 7 days on MSC. * p<0.05 vs UC; # p<0.05 vs OB; + p<0.01 vs OB+LiCl; ••p<0.01 vs. 0.5 μM Proc.</p

Procaine also promotes changes on mature osteo/odontoblasts.

<p>Procaine addition for 10 days to differentiated osteoblasts cells from MSC for 31days led to a decrease in A) alkaline phosphatase activity (*** p<0.001, ** p<0.01 vs. undifferentiated cells (UC) and ## p<0.01 vs OB cells) and B) on osteo/odontogenic genes (***p<0.001 vs UC cells and ### p<0.001 vs. OB cells). C) Calcium content and D) alizarin red staining did not change with respect to OB cells after 31 days of osteo/odontogenic stimulus and the last 10 days with procaine. Images representative of three experiments.</p

Primer sequences used for RT-PCR.

<p>Primer sequences used for RT-PCR.</p

Procaine prevents osteo/odontogenic differentiation of mesenchymal stem cells (MSC).

<p>Changes in the mRNA expression of A) early markers of oste/odontogenesis and B) specific genes of mesenchymal stem cells (MSC) were determined by RT-PCR after osteo/odontogenic differentiation (OB), OB with procaine (1 μM) (OB+Proc) during 21 days. The size and intensity of amplicon was electrophoresed on agarose gel (2%). Ribosomal 18S expression was used as housekeeping. C) Runx2 transcription factor activity was determined by a commercial TransAM^™ assay. Only OB cells showed to be significantly positive for this transcription factor (*p<0.05 vs all groups). D) Western blot for cytoplasmatic protein smooth muscle-22 alpha after 21 days of osteo/odontogenic differentiation. Images are representative of three cultures.</p

Procaine down-regulates Wnt/β- catenin pathway during osteo/odontogenesis of MSC.

<p>Osteo/odontoblast stimulus (OB) increased the mRNA expression of A) Lrp5/6 and B) Frizzled 1 and decreased the mRNA expression of C) Gsk3β (***p<0.05 vs. undifferentiated cells, UC. Treatments with procaine (OB+Proc) decreased expression of Lrp5/6 and Frizzled 1 and increased expression of Gsk3β (### p<0.001 vs OB cells and ## p<0.01 vs OB cells). D) Rat mesenchymal stem cells treated with osteo/odontoblasts stimulus (OB) or osteo/odontoblasts stimulus plus procaine (OB+Proc) were stained for β-catenin immunofluorescence (green) and counterstained with DAPI (blue) to determine β-catenin subcellular localization. Merged images of β-catenin immunofluorescence and DAPI staining are shown. Original magnification: 40x. Images were representatives of three independent experiments. E) phospho-β-catenin analysis by western blot showed that osteo/odontogenesis of MSC leads to a decrease of this protein while the presence of Procaine recovered the cytoplasmic levels of phospho β-catenin after 21 days of culture. Images are representative of three cultures. F) mRNA expression of sclerostin was decreased by osteo/odontoblast stimulus (OB) (p<0.05 vs UC) while procaine administration significantly increased the expression of this inhibitor (p<0.05 vs OB). Ribosomal 18S expression was used as housekeeping.</p

Non cytotoxic effect of Procaine.

<p>The effect of several doses of procaine (0.5, 1 and 2 μM) on cell proliferation and viability was studied onundifferentiated mesenchymal stem cells (UC) (A), MSC plus Procaine 0.5 μM (B), 1 μM (C) y 2 μM (D) for 48h, 7 and 21 days of treatment. Changes in cell proliferation and viability were also studied on MSC differentiation into osteo/odontoblasts (E) at 48h, 14d and 21d. The effects of procaine on cell proliferation and viability on these osteo/odontoblasts were studied at 0.5 μM (F), 1 μM (G) and 2 μM (H). * p<0.001 vs 48h and # p<0.001 vs 7d.</p