Implicación de la disfunción de la cadena respiratoria mitocondrial en la modulación de la respuesta inflamatoria en la artrosis en condrocitos humanos normales en cultivo : efecto de la disfunción mitocondrial en un modelo animal de inflamación articula

[Resumen] La artrosis (OA) es la patología articular más frecuente en la población, afectando a más del 70% de los mayores de 65 años. Esta enfermedad se caracteriza por la degradación progresiva del cartílago articular hialino, engrosamiento del hueso subcondral y un grado variable de inflamación sinovial. Actualmente hay un consenso general señalando a la inflamación como factor clave en su progresión, siendo las citoquinas interleuquina 1b (IL-1b) y el factor de necrosis tumoral a (TNF-a) dos de los principales mediadores implicados en esta patología. Diversos estudios ex vivo e in vitro han demostrado que los condrocitos humanos artrósicos tienen alterada la función mitocondrial. Esta disfunción mitocondrial puede afectar a varias de las vías patológicas que median en la degradación del cartílago, destacando la respuesta defectuosa del condrocito al crecimiento y a la biosíntesis de su matriz, el incremento de la apoptosis en el condrocito y la calcificación de la matriz del cartílago; así como un incremento de la respuesta inflamatoria y destructiva. En relación con el tejido sinovial, existe una asociación entre la mutagénesis mitocondrial y la respuesta inflamatoria en la artritis inflamatoria. Además, se ha observado que diversos mediadores inflamatorios presentes en el tejido artrósico, como IL-1b y TNF-a, pueden alterar la función mitocondrial tanto en el condrocito como en el sinoviocito. Por todo ello, la importancia de la mitocondria y la respuesta inflamatoria en el desarrollo de la OA es evidente. En este trabajo, se demuestra por primera vez que la disfunción mitocondrial amplifica la respuesta inflamatoria inducida por citoquinas en condrocitos humanos normales in vitro, reforzándose la hipótesis de que un daño en la función mitocondrial puede participar activamente en el fenotipo inflamatorio observado en el condrocito artrósico. Este efecto es mediado, al menos en parte, por un incremento del estrés oxidativo y de la activación del factor nuclear NF-kB, así como de la activación de las caspasas. Además, el resveratrol, una molécula anti-oxidante y anti-inflamatoria, presente en algunos alimentos como las uvas y derivados, es capaz de reducir la respuesta catabólica inducida por la sinergia entre la disfunción mitocondrial y las citoquinas; consolidándose como una molécula prometedora para fines terapéuticos y preventivos en la OA. Finalmente, en este estudio se desarrolla in vivo un modelo articular murino de daño mitocondrial, demostrándose que la disfunción mitocondrial en los tejidos articulares puede provocar la destrucción articular, al incrementar la respuesta inflamatoria.[Resumo] A artrose (OA) é a patoloxía articular mais frecuente na poboación, afectando a máis do 70% dos maiores de 65 anos. Esta enfermidade caracterízase pola degradación progresiva da cartilaxe articular hialina, engrosamento do óso subcondral e un grado variable de inflamación sinovial. Actualmente hai un consenso xeral que sinala á inflamación como factor clave na sua progresión, sendo as citoquinas interleuquina 1b (IL-1b) e o factor de necrose tumoral a (TNF-a) dous dos principiais mediadores implicados nesta patoloxía. Diversos estudos ex vivo e in vitro demostraron que os condrocitos humanos artrósicos teñen alterada a función mitocondrial. Esta disfunción mitocondrial pode afectar a varias das vías patolóxicas que median a degradación da cartilaxe, destacando a resposta defectuosa do condrocito ó crecemento e á biosíntese da matriz, o incremento da apoptose no condrocito e a calcificación da matriz da cartilaxe; así como un incremento da resposta inflamatoria e destructiva. En relación ó tecido sinovial, existe unha asociación entre a mutaxénese mitocondrial e a resposta inflamatoria na artrite inflamatoria. Ademais, observouse que diversos mediadores inflamatorios presentes no tecido artrósico, coma IL-1b e TNF-a, poden alterar a función mitocondrial tanto no condrocito coma no sinoviocito. Por todo iso, a importancia da mitocondria e a resposta inflamatoria no desenvolvemento da OA é evidente. Neste traballo, demóstrase por primeira vez que a disfunción mitocondrial amplifica a resposta inflamatoria inducida por citoquinas en condrocitos humanos normais in vitro, reforzando a hipótese de que un dano na función mitocondrial pode participar activamente no fenotipo inflamatorio observado no condrocito artrósico. Este efecto é mediado, polo menos en parte, por un incremento do estrés oxidativo e da activación do factor nuclear NF-kB, así coma da activación das caspasas. Ademais, o resveratrol, unha molécula anti-oxidante e anti-inflamatoria, presente nalguns alimentos como as uvas e derivados, é capaz de reducir a resposta catabólica inducida pola sinerxia entre a disfunción mitocondrial e as citoquinas; consolidándose como unha molécula prometedora para fins terapéuticos e preventivos na OA. Finalmente, neste estudo desenvólvese in vivo un modelo articular murino de dano mitocondrial, demostrándose que a disfunción mitocondrial nos tecidos articulares pode provocar a destrucción articular, ó incrementar a resposta inflamatoria.[Abstract] Osteoarthritis (OA) is the most common joint disease, affecting 70% of people over 65 years old. This disease is characterized by a gradual loss articular cartilage integrity, subcondral bone sclerosis and a variable grade of synovial inflammation. Currently, it is known that inflammation plays an important role in the disease progresion, and interleukin 1b (IL-1b) and tumoral necrosis factor a (TNF-a) are considered the main cytokines involved in this pathology. An increasing number of in vitro and in vivo studies have demostrated that osteoarthritic human chondrocytes present an altered mitochondrial function. This mitochondrial dysfunction may affect several pathways that have been implicated in cartilage degradation, including defective chondrocyte biosynthesis and growth responses, increased condrocyte apoptosis and matrix calcification; as well as an increment in destructive and inflammatory response. In relation to synovial tissue, an asociation between mitochondrial mutagenesis and inflammatory response has been described in inflammatory arthritis. Furthermore, diverse pro-inflamatory mediators found in the osteoarthritic condition, such as IL-1b and TNF-a, could alter mitochondrial function in the condrocyte and synoviocyte. Therefore, the role of the mitochodrion and inflammatory pathways in the pathogenesis of OA is evident. In this study, we demonstrate for the first time that mitochondrial dysfunction amplifies the inflammatory response induced by cytokines in human normal chondrocytes in vitro, supporting the hypothesis that damage in mitochondrial function could actively participate in the inflammatory phenotype observed in the osteoarthitic chondrocyte. This effect is mediated, at least partially, by an increase in oxidative stress, activation of the nuclear factor NF-kB and caspase activation. Furthermore, resveratrol, an anti-oxidant and anti-inflammatory molecule, found in certain foods such as black grapes and derivatives, is able to reduce the catabolic response induced by the synergy between mitochondrial dysfunction and cytokines. This finding confirms resveratrol as a hopeful molecule in preventive and therapeutical treatments in OA. Finally, in this thesis an in vivo murine model of articular mitochondrial damage was also developed, showing that mitochondrial dysfunction in articular tissues may trigger the joint destruction by increasing the inflammatory response

Impairment of hydrogen sulfide synthesis in chondrocytes under high glucose environment: a link between type 2 diabetes and osteoarthritis

Poster presentatio

Hydrogen sulfide biosynthesis is impaired in the osteoarthritic joint

[Abstract] Osteoarthritis (OA) is the most common form of arthritis and it is a leading cause of disability in the elderly. Its complete etiology is not known although there are several metabolic, genetic, epigenetic, and local contributing factors involved. At the moment, there is no cure for this pathology and treatment alternatives to retard or stop its progression are intensively being sought. Hydrogen sulfide (H2S) is a small gaseous molecule and is present in sulfurous mineral waters as its active component. Data from recent clinical trials shows that balneotherapy (immersion in mineral and/or thermal waters from natural springs) in sulfurous waters can improve OA symptoms, in particular, pain and function. Yet, the underlying mechanisms are poorly known. Hydrogen sulfide is also considered, with NO and CO, an endogenous signaling gasotransmitter. It is synthesized endogenously with the help of three enzymes, cystathionine gamma-lyase (CTH), cystathionine beta-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MPST). Here, the expression of these three enzymes was demonstrated by quantitative real-time polymerase chain reaction (qRT-PCR) and their protein abundance [by immunohistochemistry and Western blot (WB)] in human articular cartilage. No significant differences were found in CBS or CTH expression or abundance, but mRNA and protein levels of 3-MPST were significantly reduced in cartilage form OA donors. Also, the biosynthesis of H2S from OA cartilage, measured with a specific microelectrode, was significantly lower than in OA-free tissue. Yet, no differences were found in H2S concentration in serum from OA patients and OA-free donors. The current results suggest that reduced levels of the mitochondrial enzyme 3-MPST in OA cartilage might be, at least in part, responsible for a reduction in H2S biosynthesis in this tissue and that impaired H2S biosynthesis in the joint might be a contributing factor to OA. This could contribute to explain why exogenous supplementation of H2S, for instance with sulfurous thermal water, has positive effects in OA patients.Instituto de Salud carlos III; PI12/00329Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; RETIC-RIER-RD12/0009/0018Xunta de Galicia; IN607A 2017/1

Study of Fucoidans as Natural Biomolecules for Therapeutical Applications in Osteoarthritis

[Abstract] Osteoarthritis (OA) is the most prevalent articular chronic disease. Although, to date there is no cure for OA. Fucoidans, one of the main therapeutic components of brown algae, have emerged as promising molecules in OA treatment. However, the variability between fucoidans makes difficult the pursuit of the most suitable candidate to target specific pathological processes. By an in vitro experimental approach in chondrocytes and fibroblast-like synoviocytes, we observed that chemical composition of fucoidan, and specifically the phlorotannin content and the ratio sulfate:fucose, seems critically relevant for its biological activity. Nonetheless, other factors like concentration and molecular weight of the fucoidan may influence on its beneficial effects. Additionally, a cell-type dependent response was also detected. Thus, our results shed light on the potential use of fucoidans as natural molecules in the treatment of key pathological processes in the joint that favor the development of rheumatic disorders as OA.Xunta de Galicia; ED431G2019/0

Reduced Levels of H₂S in Diabetes-Associated Osteoarthritis Are Linked to Hyperglycaemia, Nrf-2/HO-1 Signalling Downregulation and Chondrocyte Dysfunction

[Abstract] Different findings indicate that type 2 diabetes is an independent risk factor for osteoarthritis (OA). However, the mechanisms underlying the connection between both diseases remain unclear. Changes in the balance of hydrogen sulphide (H₂S) are thought to play an important role in the pathogenesis of diabetes and its complications, although its role is still controversial. In this study, we examined the modulation of H₂S levels in serum and chondrocytes from OA diabetic (DB) and non-diabetic (non-DB) patients and in cells under glucose stress, in order to elucidate whether impairment in H₂S-mediated signalling could participate in the onset of DB-related OA. Here, we identified a reduction in H₂S synthesis in the cartilage from OA-DB patients and in cells under glucose stress, which is associated with hyperglycaemia-mediated dysregulation of chondrocyte metabolism. In addition, our results indicate that H₂S is an inductor of the Nrf-2/HO-1 signalling pathway in cartilage, but is also a downstream target of Nrf-2 transcriptional activity. Thereby, impairment of the H₂S/Nrf-2 axis under glucose stress or DB triggers chondrocyte catabolic responses, favouring the disruption of cartilage homeostasis that characterizes OA pathology. Finally, our findings highlight the benefits of the use of exogeneous sources of H₂S in the treatment of DB-OA patients, and warrant future clinical studies.This research was funded by grant PI19/01206 from the Fondo de Investigación Sanitaria, integrated in the National Plan for Scientific Program, Development, and Technological Innovation 2013–2016 and funded by the Instituto de Salud Carlos III (ISCIII)-General Subdirection of Assessment and Promotion of Research-European Regional Development Fund (FEDER) “A way of making Europe”, and also by grants ED431B 2020/55 (Grupos con Potencial de Crecemento 2020) and IN607A 2021/7 (Grupos de Referencia Competitiva) from Xunta de Galicia. The study was also supported by the Biomedical Research Network Centre (CIBER), an initiative of ISCIII. C.V.-G. thanks Xunta de Galicia for his postdoctoral contract (grant number ED481D 2017/023)Xunta de Galicia; ED431B 2020/55Xunta de Galicia; IN607A 2021/7Xunta de Galicia; ED481D 2017/02

Effect of balneotherapy in sulfurous water on an in vivo murine model of osteoarthritis

[Abstract] Osteoarthritis (OA) is a chronic joint disease that results in progressive cartilage destruction and subsequently joint dysfunction. Growing evidence indicates beneficial impact of balneological interventions in OA; however, their mechanisms of action are still unclear. Here, we evaluate the effect of balneotherapy in sulfurous water in an OA experimental model. Experimental OA was induced in Wistar rats by transection of the medial collateral ligament and removal of the medial meniscus of the left knee. Animals were randomized into three groups: non-treated (control) and balneotherapy using sulfurous water (SW) or tap water (TW). Macroscopic evaluation was performed, as well as evaluation of pain levels and analysis of motor function by rotarod test. Histopathological changes in articular cartilage and synovium were also evaluated. The presence of matrix metalloproteinase-13 (MMP-13) and oxidative damage markers was assessed by immunohistochemistry. Joint destabilization induced joint thickening, loss of joint flexion, and increased levels of pain. At day 40, animals from SW group presented lower pain levels than those from control group. Experimental OA also affected motor function. Balneotherapy in sulfur-rich water significantly improved joint mobility in relation to that in tap water. Besides, we observed that cartilage deterioration was lower in SW group than in the other two groups. Likewise, SW group showed reduced levels of MMP-13 in the cartilage. Conversely, we failed to observe any modulation on synovial inflammation. Finally, balneotherapy in sulfurous water diminished the presence of oxidative damage markers. Our results suggest the beneficial effect of balneotherapy in sulfur-rich water on an experimental model of OA, showing a reduced cartilage destruction and oxidative damage. Thus, these findings support the use of balneotherapy as a non-pharmacological treatment in OA.Instituto de Salud Carlos III; PI16/02124Xunta de Galicia; AGRUP2015/05 CICA-INIBICXunta de Galicia; IN607A 2017/11(GPC

In vitro study of the therapeutic potential of brown crude fucoidans in osteoarthritis treatment

[Abstract] Osteoarthritis, one of the most common joint degenerative pathologies, still has no cure, and current treatments, such as nonsteroidal anti-inflammatory drugs, can cause serious adverse effects when taken for a long time. Brown seaweed crude fucoidans are used for the clinical treatment of several pathologies. In this study, the therapeutical potential of these biocompounds was analyzed in primary chondrocytes and the 260TT human chondrocyte cell line. Crude fucoidan from Undaria pinnatifida (Up) and Sargassum muticum (Sm) was obtained by different extraction techniques (microwave-assisted extraction, pressurized hot-water extraction, ultrasound-assisted extraction) and chemically and structurally characterized by Fourier transform infrared spectroscopy, high-performance size-exclusion chromatography, proton nuclear magnetic resonance, and scanning electron microscopy. Once cell viability was confirmed in chondrocytes treated with crude fucoidans, we evaluated their anti-inflammatory effects, observing a significant reduction in IL-6 production stimulated by IL-1β. Findings were confirmed by analysis of IL-6 and IL-8 gene expression, although only fucoidans from Up achieved a statistically significant reduction. Besides this, the antioxidant capacity of crude fucoidans was observed through the upregulation of Nrf-2 levels and the expression of its transcriptional target genes HO-1 and SOD-2, with compounds from Up again showing a more consistent effect. However, no evidence was found that crude fucoidans modulate senescence, as they failed to reduced β-galactosidase activity, cell proliferation, or IL-6 production in chondrocytes stimulated with etoposide. Thus, the findings of this research seem to indicate that the tested crude fucoidans are capable of partially alleviating OA-associated inflammation and oxidative stress, but fail to attenuate chondrocyte senescence

Long-term effects of hydrogen sulfide on the anabolic-catabolic balance of articular cartilage in vitro

[Abstract] Healthy cartilage maintenance relies on an equilibrium among the anabolic and catabolic processes in chondrocytes. With the onset of osteoarthritis (OA), increased interleukin (IL)-1β levels induce an inhibition of the synthesis of extracellular matrix (ECM) proteins, as well as an increase in proteases. This eventually leads to a predominance of the catabolic phenotype and the progressive loss of articular cartilage. Hydrogen sulfide (H2S) is a small gaseous molecule recognized as the third endogenous gasotransmitter. When administered exogenously, it has shown anti-inflammatory and anti-catabolic properties in several in vitro and in vivo models. Here, OA cartilage disks were co-cultured in vitro with IL-1β (5 ng/ml) and NaSH or GYY4137 (200 or 1000 μM) for 21 days. The ability of these two H2S-producing compounds to avoid long term extracellular matrix (ECM) destruction was evaluated. We used a glycosaminoglycan (GAG) quantification kit histology and immunohistochemistry (IHC) to evaluate matrix proteins degradation and matrix metalloproteinases (MMP) abundance. Through the GAGs quantification assay, safranin O (S-O) and toluidine blue (TB) stains, and keratan/chondroitin sulfate (KS/ChS) IHCs it was shown that co-stimulation with H2S-forming reagents effectively avoided GAGs destruction. Both Masson's trichrome (MT) stain and collagen (col) type II IHC, as well as aggrecan (agg) IHC demonstrated that not only were these proteins protected but even promoted, their abundance being higher than in the basal condition. Further, stains also demonstrated that positivity in the inter-territorial and intra-cellular for the different matrix components were rescued, suggesting that NaSH and GYY4137 might also have pro-anabolic effects. In addition, a clear protective effect against the increased MMPs levels was seen, since increased MMP3 and 13 levels were subsequently reduced with the co-stimulation with sulfide compounds. In general, GYY4137 was more effective than NaSH, and increasing the dose improved the results. This study demonstrates that H2S anti-catabolic effects, which had been previously proven in short-term (24–48 h) in vitro cellular models, are maintained over time directly in OA cartilage tissue.Instituto de Salud Carlos III; PI12/0032

Study of hydrogen sulfide biosynthesis in synovial tissue from diabetes-associated osteoarthritis and its influence on macrophage phenotype and abundance

[Abstract] Type 2 diabetes (DB) is an independent risk factor for osteoarthritis (OA). However, the mechanisms underlying the connection between both diseases remain unclear. Synovial macrophages from OA patients with DB present a marked pro-inflammatory phenotype. Since hydrogen sulphide (H2S) has been previously described to be involved in macrophage polarization, in this study we examined H2S biosynthesis in synovial tissue from OA patients with DB, observing a reduction of H2S-synthetizing enzymes in this subset of individuals. To elucidate these findings, we detected that differentiated TPH-1 cells to macrophages exposed to high levels of glucose presented a lower expression of H2S-synthetizing enzymes and an increased inflammatory response to LPS, showing upregulated expression of markers associated with M1 phenotype (i.e., CD11c, CD86, iNOS, and IL-6) and reduced levels of those related to M2 fate (CD206 and CD163). The co-treatment of the cells with a slow-releasing H2S donor, GYY-4137, attenuated the expression of M1 markers, but failed to modulate the levels of M2 indicators. GYY-4137 also reduced HIF-1α expression and upregulated the protein levels of HO-1, suggesting their involvement in the anti-inflammatory effects of H2S induction. In addition, we observed that intraarticular administration of H2S donor attenuated synovial abundance of CD68+ cells, mainly macrophages, in an in vivo model of OA. Taken together, the findings of this study seem to reinforce the key role of H2S in the M1-like polarization of synovial macrophages associated to OA and specifically its metabolic phenotype, opening new therapeutic perspectives in the management of this pathology

Intraarticular Administration Effect of Hydrogen Sulfide on an In Vivo Rat Model of Osteoarthritis

[Abstract] Osteoarthritis (OA) is the most common articular chronic disease. However, its current treatment is limited and mostly symptomatic. Hydrogen sulfide (H2S) is an endogenous gas with recognized physiological activities. The purpose here was to evaluate the effects of the intraarticular administration of a slow-releasing H2S compound (GYY-4137) on an OA experimental model. OA was induced in Wistar rats by the transection of medial collateral ligament and the removal of the medial meniscus of the left joint. The animals were randomized into three groups: non-treated and intraarticularly injected with saline or GYY-4137. Joint destabilization induced articular thickening (≈5% increment), the loss of joint mobility and flexion (≈12-degree angle), and increased levels of pain (≈1.5 points on a scale of 0 to 3). Animals treated with GYY-4137 presented improved motor function of the joint, as well as lower pain levels (≈75% recovery). We also observed that cartilage deterioration was attenuated in the GYY-4137 group (≈30% compared with the saline group). Likewise, these animals showed a reduced presence of pro-inflammatory mediators (cyclooxygenase-2, inducible nitric oxide synthase, and metalloproteinase-13) and lower oxidative damage in the cartilage. The increment of the nuclear factor-erythroid 2-related factor 2 (Nrf-2) levels and Nrf-2-regulated gene expression (≈30%) in the GYY-4137 group seem to be underlying its chondroprotective effects. Our results suggest the beneficial impact of the intraarticular administration of H2S on experimental OA, showing a reduced cartilage destruction and oxidative damage, and supporting the use of slow H2S-producing molecules as a complementary treatment in OA.Instituto de Salud Carlos III; PI16/02124Xunta de Galicia; AGRUP2015/05 CICA-INIBICXunta de Galicia; IN607A 2017/1