Osteogenic Potential of Monosodium Urate Crystals in Synovial Mesenchymal Stem Cells

Background and Objectives: Deposits of monosodium urate (MSU) crystals due to increased levels of uric acid (UA) have been associated with bone formation and erosion, mainly in patients with chronic gout. The synovial membrane (SM) comprises several types of cells, including mesenchymal stem cells (SM-MSCs); however, it is unknown whether UA and MSU induce osteogenesis through SM-MSCs. Materials and Methods: Cultures of SM were immunotyped with CD44, CD69, CD90, CD166, CD105, CD34, and CD45 to identify MSCs. CD90+ cells were isolated by immunomagnetic separation (MACS), colony-forming units (CFU) were identified, and the cells were exposed to UA (3, 6.8, and 9 mg/dL) and MSU crystals (1, 5, and 10 μg/mL) for 3 weeks, and cellular morphological changes were evaluated. IL-1β and IL-6 were determined by ELISA, mineralization was assessed by alizarin red, and the expression of Runx2 was assessed by Western blot. Results: Cells derived from SM and after immunomagnetic separation were positive for CD90 (53 ± 8%) and CD105 (52 ± 18%) antigens, with 53 ± 5 CFU identified. Long-term exposure to SM-MSCs by UA and MSU crystals did not cause morphological damage or affect cell viability, nor were indicators of inflammation detected. Mineralization was observed at doses of 6.8 mg/dL UA and 5 μg/mL MSU crystals; however, the differences were not significant with respect to the control. The highest dose of MSU crystals (10 μg/mL) induced significant Runx2 expression with respect to the control (1.4 times greater) and SM-MSCs cultured in the osteogenic medium. Conclusions: MSU crystals may modulate osteogenic differentiation of SM-MSCs through an increase in Runx2

Time course of EMSAs of HSF1 before (T0), immediately after heat shock (HS) and 1 (1 h) and 2 (2 h) hours after recovery at 37°C from PBMCs.

<p>Nuclear extract protein was obtained and EMSA was performed with 10 µg of protein as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065449#s4" target="_blank">Materials and Methods</a>. A) Nuclear extracts from PBMCs from control (Control) or ASA challenged rats (ASA 1<b> </b>h) were analyzed at the indicated time points. B) Nuclear extracts from PBMCs from control animals received the indicated treatments <i>in vitro</i> 1 h before heat shock. DNA/protein complexes are labeled as: I and II. Histograms represent the relative amount of complex I under the different experimental conditions, normalized against control obtained at T0, both <i>in vivo</i> and <i>in vitro</i>.</p

Time course of changes in HSP-72 expression before (T0), immediately after heat shock (HS) and 1 (1 h) and 2 (2 h) hours after recovery at 37°C in peripheral blood mononuclear cells (PBMCs) from untreated rats incubated with or without the combination ASA (10 µM)+H₂O₂ (5 µM).

<p>A) Western blots of HSP-72 from rat PBMCs (100 µg of total protein) incubated with (+) or without (-) the indicated treatments for 1 h before heat shock, β-actin was used as loading control. Protein extracts were obtained before <i>in vitro</i> incubation (T0), or immediately after Heat shock (HS) or (1<b> </b>h) and 2 (2<b> </b>h) hours after recovery at 37°C. B) Histogram of the average signals expressed as Arbitrary units (AU) = OD for HSP-72/OD for β-actin from three independent experiments. *Comparison of HSP-72 content immediately after heat shock between cells without any treatment an those incubated with the combination ASA+H₂O₂ (<i>n</i> = 3) (<i>p</i><0.001). **Comparison of cells incubated 1 h after heat shock without any treatment an those incubated with the combination ASA+H₂O₂ (<i>n</i> = 3) (<i>p</i><0.001). C) RT-PCR of HSP-72 mRNA from PBMCs treated <i>in vitro</i> with the combination ASA+H₂O₂ following heat shock. Total RNA was obtained immediately after heat shock. Products of RT-PCR were resolve in agarose gels.</p

Time course of changes in HSP-72 expression before (T0), immediately after heat shock (HS) and 1 (1 h) and 2 (2 h) hours after recovery at 37°C in peripheral blood mononuclear cells (PBMCs) from rats, treated with or without acetylsalicylic acid (ASA 45 mg/kg of body weight).

<p>Cells were isolated 1, 2; 3, or 5 h after administration of ASA and subjected to heat shock protocol. Cells from untreated animals served as controls. 100 µg of total protein was placed per lane. A) Representative Western blots corresponding to cells from control animals or from rats challenged with ASA for 1, 2, 3, or 5 h. B) Histogram of the average signals expressed as Arbitrary Units (AU = OD for HSP-72/OD for β-actin) from three independent experiments. *Comparison of HSP-72 content immediately after heat shock between cells from ASA-challenged rats (for 1, 2, 3, or 5 h) or without it (control) (<i>n</i> = 3) (<i>p</i><0.05). **A similar comparison 1 h after heat shock (<i>n</i> = 3) (<i>p</i><0.05). <i>t</i>-student test was used. C) RT-PCR for HSP-72 mRNA content in PBMCs from rats with or without ASA treatment. PBMCs were obtained 1 h after ASA challenged and subjected to in vitro to heat shock. Total RNA was obtained immediately after heat shock. Products of RT-PCR were resolve in agarose gels. The numbers indicate the normalized band intensity (HSP-72/GAPDH).</p

Dual regulation of energy metabolism by p53 in human cervix and breast cancer cells

AbstractThe role of p53 as modulator of OxPhos and glycolysis was analyzed in HeLa-L (cells containing negligible p53 protein levels) and HeLa-H (p53-overexpressing) human cervix cancer cells under normoxia and hypoxia. In normoxia, functional p53, mitochondrial enzyme contents, mitochondrial electrical potential (ΔΨm) and OxPhos flux increased in HeLa-H vs. HeLa-L cells; whereas their glycolytic enzyme contents and glycolysis flux were unchanged. OxPhos provided more than 70% of the cellular ATP and proliferation was abolished by anti-mitochondrial drugs in HeLa-H cells. In hypoxia, both cell proliferations were suppressed, but HeLa-H cells exhibited a significant decrease in OxPhos protein contents, ΔΨm and OxPhos flux. Although glycolytic function was also diminished vs. HeLa-L cells in hypoxia, glycolysis provided more than 60% of cellular ATP in HeLa-H cells. The energy metabolism phenotype of HeLa-H cells was reverted to that of HeLa-L cells by incubating with pifithrin-α, a p53-inhibitor. In normoxia, the energy metabolism phenotype of breast cancer MCF-7 cells was similar to that of HeLa-H cells, whereas p53shRNAMCF-7 cells resembled the HeLa-L cell phenotype. In hypoxia, autophagy proteins and lysosomes contents increased 2–5 times in HeLa-H cells suggesting mitophagy activation. These results indicated that under normoxia p53 up-regulated OxPhos without affecting glycolysis, whereas under hypoxia, p53 down-regulated both OxPhos (severely) and glycolysis (weakly). These p53 effects appeared mediated by the formation of p53-HIF-1α complexes. Therefore, p53 exerts a dual and contrasting regulatory role on cancer energy metabolism, depending on the O2 level

Molecular structure of quinolinic acid (QUIN).

<p>QUIN and its conversion to a dianionic stucture (pK1 = 2.43 and pK2 = 4.78) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120221#pone.0120221.ref040" target="_blank">40</a>] at pH 7.4. Each structure was built up with the program ACD/ChemSketch Freeware (<a href="http://www.acdlabs.com/resources/freeware/chemsketch/" target="_blank">http://www.acdlabs.com/resources/freeware/chemsketch/</a>).</p

Additional file 5: of Monosodium urate crystals induce oxidative stress in human synoviocytes

Characterization of cell populations. Synoviocytes culture marked with anti-CD166 and anti-CD14. Each bar shows the average ± standard deviation of three independent experiments from different patients (n = 5); *P < 0.05. (DOCX 26 kb

Additional file 1: Table S1. of Monosodium urate crystals induce oxidative stress in human synoviocytes

Primers used for qRT-PCR gene analysis. (DOCX 14 kb