Monosodium urate crystals induce oxidative stress in human synoviocytes

Yessica Zamudio-Cuevas, Karina Martínez-Flores, Javier Fernández-Torres, Yahir A Loissell-Baltazar, Daniel Medina-Luna, Ambar López-Macay, Javier Camacho-Galindo, Cristina Hernández-Díaz, Mónica G Santamaría-Olmedo, Edgar Oliver López-Villegas, Francesca Oliviero, Anna Scanu, Jorge Francisco Cerna-Cortés, Marwin Gutierrez, Carlos Pineda, Alberto López-Reyes, Yessica Zamudio-Cuevas, Karina Martínez-Flores, Javier Fernández-Torres, Yahir A Loissell-Baltazar, Daniel Medina-Luna, Ambar López-Macay, Javier Camacho-Galindo, Cristina Hernández-Díaz, Mónica G Santamaría-Olmedo, Edgar Oliver López-Villegas, Francesca Oliviero, Anna Scanu, Jorge Francisco Cerna-Cortés, Marwin Gutierrez, Carlos Pineda, Alberto López-Reyes

Abstract

Background: Gout is the most common inflammatory arthropathy of metabolic origin and it is characterized by intense inflammation, the underlying mechanisms of which are unknown. The aim of this study was to evaluate the oxidative stress in human fibroblast-like synoviocytes (FLS) exposed to monosodium urate (MSU) crystals, which trigger an inflammatory process.

Methods: Human FLS isolated from synovial tissue explants were stimulated with MSU crystals (75 μg/mL) for 24 h. Cellular viability was evaluated by crystal violet staining, apoptosis was assessed using Annexin V, and the cellular content of reactive oxygen species (ROS) and nitrogen species (RNS) (O2 (-), H2O2, NO) was assessed with image-based cytometry and fluorometric methods. In order to determine protein oxidation levels, protein carbonyls were detected through oxyblot analysis, and cell ultrastructural changes were assessed by transmission electron microscopy.

Results: The viability of FLS exposed to MSU crystals decreased by 30 % (P < 0.05), while apoptosis increased by 42 % (P = 0.01). FLS stimulated with MSU crystals exhibited a 2.1-fold increase in H2O2 content and a 1.5-fold increase in O2 (-) and NO levels. Oxyblots revealed that the spots obtained from FLS protein lysates exposed to MSU crystals exhibited protein carbonyl immunoreactivity, which reflects the presence of oxidatively modified proteins. Concomitantly, MSU crystals triggered the induction of changes in the morphostructure of FLS, such as the thickening and discontinuity of the endoplasmic reticulum, and the formation of vacuoles and misfolded glycoproteins.

Conclusions: Our results prove that MSU crystals induce the release of ROS and RNS in FLS, subsequently oxidizing proteins and altering the cellular oxidative state of the endoplasmic reticulum, which results in FLS apoptosis.

Keywords: Gout; Monosodium urate crystals; Oxidative stress; Synoviocytes.

Figures

Fig. 1
Fig. 1
Cellular response to the presence of monosodium urate (MSU) crystals. a Cell viability after a 24 h treatment. b Cell morphological changes after MSU crystal exposure. The arrows indicate the intracellular vacuoles of MSU crystals. c Apoptosis is revealed by Annexin V detection (yellow arrows) in synoviocytes exposed to MSU crystals and H2O2 (100 μM). Additionally, columns show quantification of the apoptotic cells by flow cytometry. Values are expressed as the mean ± standard deviation *P < 0.05 vs control
Fig. 2
Fig. 2
Monosodium urate (MSU) crystals increase reactive oxygen species (ROS) in synoviocytes. Arrows indicate intracellular H2O2 formation, which is revealed by DCFH oxidation (green fluorescence) in untreated fibroblast-like synoviocytes (FLS) (a); FLS treated with MSU crystals at 24 h (b), and FLS treated with H2O2 at 30 minutes (c). Arrows indicate O2 - intracellular production by oxidation of dihydroethidium (DHE) (red fluorescence) in untreated FLS (d); FLS treated with MSU crystals (e), and FLS treated with H2O2 (f). Bars show quantification of DCFH and DHE fluorescence: data are reported as units of arbitrary fluorescence (UAF) (g). Values are expressed as the mean ± standard deviation; *P < 0.05 vs control
Fig. 3
Fig. 3
Nitric oxide (NO) production in synoviocytes. a Detection of NO in untreated fibroblast-like synoviocytes (FLS). b FLS treated with monosodium urate (MSU) crystals. c FLS treated with H2O2. Arrows indicate fluorescence produced by intracellular NO. d Bars show NO quantification by Tali image-based cytometer. Values are expressed as the mean ± standard deviation; *P < 0.05 vs control
Fig. 4
Fig. 4
Oxidized proteins assay. a Representative oxyblot of fibroblast-like synoviocytes (FLS) proteins from control group (line 1); FLS proteins exposed to monosodium urate (MSU) crystals (line 2); and FLS proteins from the positive control sample (line 3). b Oxidation scan with fluorescence detector. Results are representative of independent experiments with cells from different patients
Fig. 5
Fig. 5
Ultrastructural changes in synoviocytes. A Ultrastructure of an untreated fibroblast-like synoviocytes (FLS). a Magnified view of the section is indicated by a black box showing the nucleus (N), endoplasmic reticulum (ER) and vacuoles (V) highlighted with arrows. B FLS treated with monosodium urate (MSU) crystals at 75 μg/mL exhibiting N, swollen vesicular structures of different sizes, and MSU crystal cavity. b A high-magnification image showing misfolded proteins (MP) aggregates and ER indicated with arrows. C FLS treated with H2O2 at 100 μM showing N. c A magnified view of the section is indicated by a black box showing MP aggregates, ER and N. Results are representative of one of five separate experiments with FLS from different patients

References

    1. Choi HK, Niu J, Neogi T, Chen CA, Chaisson C, Hunter D, et al. Nocturnal risk of gout attacks. Arthritis Rheumatol. 2015;67:555–562. doi: 10.1002/art.38917.
    1. García-Méndez S, Arreguín-Reyes R, López-López O, Vázquez MJ. Frecuencia de la gota según la percepción de los médicos en México. Reumatol Clin. 2014;10:197–8. doi: 10.1016/j.reuma.2013.06.003.
    1. Cronstein BN, Terkeltaub R. The inflammatory process of gout and its treatment. Arthritis Res Ther. 2006;8 Suppl 1:S3. doi: 10.1186/ar1908.
    1. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschoop J. Gout associated uric acid crystals activate the NALP3 inflammasome. Nature. 2006;440:237–41. doi: 10.1038/nature04516.
    1. Stamp LK, Turner R, Khalilova IS, Zhang M, Drake J, Forbes LV, et al. Myeloperoxidase and oxidation of uric acid in gout: implications for the clinical consequences of hyperuricaemia. Rheumatology (Oxford) 2014;53:1958–65. doi: 10.1093/rheumatology/keu218.
    1. Kim SK, Choe JY, Park KY. Rebamipide suppresses monosodium urate crystal-induced interleukin-1β production through regulation of oxidative stress and caspase-1 in THP-1 cells. Inflammation. 2016;39:473–82. doi: 10.1007/s10753-015-0271-5.
    1. Pouliot M, James MJ, McColl SR, Naccache PH, Cleland LG. Monosodium urate microcrystals induce cyclooxygenase-2 in human monocytes. Blood. 2012;91:1769–76.
    1. So A, Thorens B. Uric acid transport and disease. Sci Med J Clin Invest. 2010;120:1791–9. doi: 10.1172/JCI42344.
    1. Zheng SC, Zhu XX, Xue Y, Zhang LH, Zou HJ, Qiu JH, et al. Role of the NLRP3 inflammasome in the transient release of IL-1β induced by monosodium urate crystals in human fibroblast-like synoviocytes. J Inflamm (Lond) 2015;12:30. doi: 10.1186/s12950-015-0070-7.
    1. Scanu A, Oliviero F, Gruaz L, Sfriso P, Pozzuoli A, Frezzato F, et al. High-density lipoproteins downregulate CCL2 production in human fibroblast-like synoviocytes stimulated by urate crystals. Arthritis Res Ther. 2010;2:R12.
    1. Trevisan G, Hoffmeister C, Rossato MF, Oliveira SM, Silva MA, Silva CR, et al. TRPA1 receptor stimulation by hydrogen peroxide is critical to trigger hyperalgesia and inflammation in a model of acute gout. Free Radic Biol Med. 2014;72:200–9. doi: 10.1016/j.freeradbiomed.2014.04.021.
    1. Denko CW, Whitehouse MW. Experimental inflammation induced by natural occurring microcrystalline calcium salts. J Rheumatol. 1976;3:54–62.
    1. Paul H, Reginato AJ, Schumacher HR. Morphological characteristics of monosodium urate: transmission electron microscopic study of intact natural and synthetic crystals. Ann Rheum Dis. 1983;42:75–81. doi: 10.1136/ard.42.1.75.
    1. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–9. doi: 10.1016/0003-2697(87)90021-2.
    1. Serratos IN, Castellanos P, Pastor N, Millán-Pacheco C, Rembao D, Pérez-Montfort R, et al. Modeling the Interaction between quinolinate and the receptor for advanced glycation end products (RAGE): relevance for early neuropathological processes. PLoS One. 2015;10(3):e0120221. doi: 10.1371/journal.pone.0120221.
    1. Landa-Solís C, Granados-Montiel J, Olivos-Meza A, Ortega-Sánchez C, Cruz-Lemini M, Hernández-Flores C, et al. Cryopreserved CD90+ cells obtained from mobilized peripheral blood in sheep: a new source of mesenchymal stem cells for preclinical applications. Cell Tissue Bank. 2016;17(1):137–45. doi: 10.1007/s10561-015-9526-5.
    1. Flick DA, Gifford G. Comparison of in vitro cell cytotoxic assays for tumor necrosis factor. J Immunol Methods. 1984;68:167–75. doi: 10.1016/0022-1759(84)90147-9.
    1. Zamudio-Cuevas Y, Díaz-Sobac R, Vázquez-Luna A, Landa-Solís C, Cruz-Ramos M, Santamaría-Olmedo M, et al. The antioxidant activity of soursop decreases the expression of a member of the NADPH oxidase family. Food Funct. 2014;5(2):303–9. doi: 10.1039/C3FO60135H.
    1. Stankovíc A, Front P, Barbara A, Mitrovíc DR. Tophus‐derived monosodium urate monohydrate crystals are biologically much more active than synthetic counterpart. Rheumatol Int. 1991;10:221–6. doi: 10.1007/BF02274882.
    1. Tudan C, Jackson JK, Blanis L, Pelech SL, Burt HM. Inhibition of TNF-alpha-induced neutrophil apoptosis by crystals of calcium pyrophosphate dihydrate is mediated by the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways up-stream of caspase 3. J Immunol. 2000;165:5798–806. doi: 10.4049/jimmunol.165.10.5798.
    1. Chhana A, Callon KE, Pool B, Naot D, Watson M, Gamble GD, et al. Monosodium urate monohydrate crystals inhibit osteoblast viability and function: implications for development of bone erosion in gout. Ann Rheum Dis. 2011;70:1684–91. doi: 10.1136/ard.2010.144774.
    1. Choe JY, Park KY, Kim SK. Oxidative stress by monosodium urate crystals promotes renal cell apoptosis through mitochondrial caspase-dependent pathway in human embryonic kidney 293 cells: mechanism for urate-induced nephropathy. Apoptosis. 2015;20:38–49. doi: 10.1007/s10495-014-1057-1.
    1. Malemud CJ, Sun Y, Pearlman E, Ginley NM, Awadallah A, Wisler BA, et al. Monosodium urate and tumor necrosis factor-α increase apoptosis in human chondrocyte cultures. Rheumatology (Sunnyvale) 2012;2:113. doi: 10.4172/2161-1149.1000113.
    1. David-Raoudi M, Deschrevel B, Leclercq S, Galéra P, Boumediene K, Pujol JP. Chondroitin sulfate increases hyaluronan production by human synoviocytes through differential regulation of hyaluronan synthases: Role of p38 and Akt. Arthritis Rheum. 2009;60:760–70. doi: 10.1002/art.24302.
    1. Cillero-Pastor B, Martin MA, Arenas J, López-Armada MJ, Blanco FJ. Effect of nitric oxide on mitochondrial activity of human synovial cells. BMC Musculoskelet Dis. 2011;12:42. doi: 10.1186/1471-2474-12-42.
    1. Van’t Hof RJ, Hocking L, Wright PK, Ralston SH. Nitric oxide is a mediator of apoptosis in the rheumatoid joint. Rheumatology (Oxford) 2000;39:1004–8. doi: 10.1093/rheumatology/39.9.1004.
    1. Jhang JJ, Cheng YT, Ho CY, Yen GC. Monosodium urate crystals trigger Nrf2- and heme oxygenase-1-dependent inflammation in THP-1 cells. Cell Mol Immunol. 2015;12:424–34. doi: 10.1038/cmi.2014.65.
    1. Chenevier-Gobeaux C, Lemarechal H, Bonnefont-Rousselot D, Poiraudeau S, Ekindjian OG, Borderie D. Superoxide production and NADPH oxidase expression in human rheumatoid synovial cells: regulation by interleukin-1beta and tumor necrosis factor-alpha. Inflamm Res. 2006;55:483–90. doi: 10.1007/s00011-006-6036-8.
    1. Zheng S, Zhong ZM, Qin S, Chen GX, Wu Q, Zeng JH, et al. Advanced oxidation protein products induce inflammatory response in fibroblast like synoviocytes through NADPH oxidase dependent activation of NF-κB. Cell Physiol Biochem. 2013;32:972–85. doi: 10.1159/000354500.
    1. Fedorova M, Kuleva N, Hoffmann R. Reversible and irreversible modifications of skeletal muscle proteins in a rat model of acute oxidative stress. Biochim Biophys Acta. 2009;1792(12):1185–93. doi: 10.1016/j.bbadis.2009.09.011.
    1. Dalle-Donne I, Rossi R, Giustarini D, Milzania A, Colombo R. Protein carbonyl groups as biomarkers of oxidative stress. 2003. Clin Chim Acta. 2003;329:23–38. doi: 10.1016/S0009-8981(03)00003-2.
    1. Merry P, Winyard PG, Morris CJ, Grootveld M, Blake DR. Oxygen free radicals, inflammation, and synovitis: and synovitis: the current status. Ann Rheum Dis. 1989;48:864–70. doi: 10.1136/ard.48.10.864.
    1. Dai M, Wei W, Shen YX, Zheng YQ. Glucosides of Chaenomeles speciose remit rat adjuvant arthritis by inhibiting synoviocyte activities. Acta Pharmacol Sin. 2003;24:1161–6.
    1. Butoescu N, Seemayer CA, Foti M, Jordan O, Doelker E. Dexamethasone-containing PLGA superparamagnetic microparticles as carriers for the local treatment of arthritis. Biomaterials. 2009;30:1772–80. doi: 10.1016/j.biomaterials.2008.12.017.
    1. Shin HY, Oh JM, Kim YS. The functional role of prion protein (PrPC) on autophagy. Pathogens. 2013;2:436–45. doi: 10.3390/pathogens2030436.
    1. Betteridge DJ. What is oxidative stress? Metabolism. 2000;49:3–8. doi: 10.1016/S0026-0495(00)80077-3.

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