Mitochondrial respiratory chain dysfunction modulates metalloproteases -1, -3 and -13 in human normal chondrocytes in culture

Background: Mitochondrion has an important role in the osteoarthritis (OA) pathology. We have previously demonstrated that the alteration of the mitochondrial respiratory chain (MRC) contributes to the inflammatory response of the chondrocyte. However its implication in the process of cartilage destruction is not well understood yet. In this study we have investigated the relationship between the MRC dysfunction and the regulation of metalloproteases (MMPs) in human normal chondrocytes in culture. Methods: Human normal chondrocytes were isolated from human knees obtained form autopsies of donors without previous history of rheumatic disease. Rotenone, 3-Nitropropionic acid (NPA), Antimycin A (AA), Sodium azide and Oligomycin were used to inhibit the activity of the mitochondrial complexes I, II, III, IV and V respectively. The mRNA expression of MMPs -1, -3 and -13 was studied by real time PCR. The intracellular presence of MMP proteins was evaluated by western blot. The liberation of these proteins to the extracellular media was evaluated by ELISA. The presence of proteoglycans in tissue was performed with tolouidin blue and safranin/fast green. Immunohistochemistry was used for evaluating MMPs on tissue. Results: Firstly, cells were treated with the inhibitors of the MRC for 24 hours and mRNA expression was evaluated. An up regulation of MMP-1 and -3 mRNA levels was observed after the treatment with Oligomycin 5 and 100 μg/ml (inhibitor of the complex V) for 24 hours. MMP-13 mRNA expression was reduced after the incubation with AA 20 and 60 μg/ml (inhibitor of complex III) and Oligomycin. Results were validated at protein level observing an increase in the intracellular levels of MMP-1 and -3 after Oligomycin 25 μg/ml stimulation [(15.20±8.46 and 4.59±1.83 vs. basal=1, respectively (n=4; *P<0.05)]. However, AA and Oligomycin reduced the intracellular levels of the MMP-13 protein (0.70±0.16 and 0.3±0.24, respectively vs. basal=1). In order to know whether the MRC dysfunction had an effect on the liberation of MMPs, their levels were evaluated in the supernatants. After 36 hours of stimulation, values were: MMP-1=18.06±10.35 with Oligomycin 25 μg/ml vs. basal=1, and MMP-3=8.49±4.32 with Oligomycin 5 μg/ml vs. basal=1 (n=5; *P<0.05). MMP-13 levels in the supernatants were reduced after AA 60 μg/ml treatment (0.50±0.13 vs. basal=1) and Oligomycin 25 μg/ml (0.41±0.14 vs. basal=1); (n=5; *P<0.05). The treatment of explants with Oligomycin, showed an increase in the positivity of MMP-1 and -3. Explants stimulated with AA or Oligomycin revealed a decrease in MMP-13 expression. Proteoglycan staining demonstrated a reduction of proteoglycan levels in the tissues treated with Oligomycin. Conclusions: These results reveal that MRC dysfunction modulates the MMPs expression in human normal chondrocytes demonstrating its role in the regulation of the cartilage destruction

Hsp90 beta inhibition modulates nitric oxide production and nitric oxide-induced apoptosis in human chondrocytes

Background: Hsp90 beta is a member of the Hsp90 family of protein chaperones. This family plays essential roles in the folding, maturation and activity of many proteins that are involved in signal transduction and transcriptional regulation. The role of this protein in chondrocytes is not well understood, although its increase in osteoarthritic cells has been reported. The present study aimed to explore the role of Hsp90 beta in key aspects of OA pathogenesis. Methods: Human OA chondrocytes were isolated from cartilage obtained from patients undergoing joint replacement surgery, and primary cultured. Cells were stimulated with proinflammatory cytokines (IL-1 beta or TNF-alpha) and nitric oxide donors (NOC-12 or SNP). For Hsp90 beta inhibition, two different chemical inhibitors (Geldanamycin and Novobiocin) were employed, or siRNA transfection procedures were carried out. Gene expression was determined by real-time PCR, apoptosis was quantified by flow cytometry and ELISA, and nitric oxide (NO) production was evaluated by the Griess method. Indirect immunofluorescence assays were performed to evaluate the presence of Hsp90 beta in stimulated cells. Results: Hsp90 beta was found to be increased by proinflammatory cytokines. Inhibition of Hsp90 beta by the chemicals Geldanamycin (GA) and Novobiocin (NB) caused a dose-dependent decrease of the NO production induced by IL-1 beta in chondrocytes, up to basal levels. Immunofluorescence analyses demonstrate that the NO donors NOC-12 and SNP also increased Hsp90 beta. Chemical inhibition or specific gene silencing of this chaperone reduced the DNA condensation and fragmentation, typical of death by apoptosis, that is induced by NO donors in chondrocytes. Conclusions: The present results show how Hsp90 beta modulates NO production and NO-mediated cellular death in human OA chondrocytes

Optical Biomarkers for the Diagnosis of Osteoarthritis through Raman Spectroscopy: Radiological and Biochemical Validation Using Ex Vivo Human Cartilage Samples

[Abstract] Osteoarthritis (OA) is the most common rheumatic disease, characterized by progressive articular cartilage degradation. Raman spectroscopy (RS) has been recently proposed as a label-free tool to detect molecular changes in musculoskeletal tissues. We used cartilage samples derived from human femoral heads to perform an ex vivo study of different Raman signals and ratios, related to major and minor molecular components of articular cartilage, hereby proposed as candidate optical biomarkers for OA. Validation was performed against the radiological Kellgren–Lawrence (K-L) grading system, as a gold standard, and cross-validated against sulfated glycosaminoglycans (sGAGs) and total collagens (Hyp) biochemical contents. Our results showed a significant decrease in sGAGs (SGAGs, A1063 cm−1/A1004 cm−1) and proteoglycans (PGs, A1375 cm−1/A1004 cm−1) and a significant increase in collagen disorganization (ColD/F, A1245 cm−1/A1270 cm−1), with OA severity. These were correlated with sGAGs or Hyp contents, respectively. Moreover, the SGAGs/HA ratio (A1063 cm−1/A960 cm−1), representing a functional matrix, rich in proteoglycans, to a mineralized matrix-hydroxyapatite (HA), was significantly lower in OA cartilage (K-L I vs. III–IV, p < 0.05), whilst the mineralized to collagenous matrix ratio (HA/Col, A960 cm−1/A920 cm−1) increased, being correlated with K-L. OA samples showed signs of tissue mineralization, supported by the presence of calcium crystals-related signals, such as phosphate, carbonate, and calcium pyrophosphate dihydrate (MGP, A960 cm−1/A1004 cm−1, MGC, A1070 cm−1/A1004 cm−1 and A1050 cm−1/A1004 cm−1). Finally, we observed an increase in lipids ratio (IL, A1450 cm−1/A1670 cm−1) with OA severity. As a conclusion, we have described the molecular fingerprint of hip cartilage, validating a panel of optical biomarkers and the potential of RS as a complementary diagnostic tool for OA.Xunta de Galicia; ED431E 2018/03Xunta de Galicia; IN607A2017/1

Characterization of microRNA expression profiles in normal and osteoarthritic human chondrocytes

[Abstract] Background. Osteoarthritis (OA) is a multifactorial disease characterized by destruction of the articular cartilage due to environmental, mechanical and genetic components. The genetics of OA is complex and is not completely understood. Recent works have demonstrated the importance of microRNAs (miRNAs) in cartilage function. MiRNAs are a class of small noncoding RNAs that regulate gene expression and are involved in different cellular process: apoptosis, proliferation, development, glucose and lipid metabolism. The aim of this study was to identify and characterize the expression profile of miRNAs in normal and OA chondrocytes and to determine their role in the OA. Methods. Chondrocytes were moved to aggregate culture and evaluated using histological and qPCR techniques. miRNAs were isolated and analyzed using the Agilent Human miRNA Microarray. Results. Of the 723 miRNAs analyzed, 7 miRNAs showed a statistically significant differential expression. Amongst these 7 human miRNAs, 1 was up-regulated in OA chondrocytes (hsa-miR-483-5p) and 6 were up-regulated in normal chondrocytes (hsa-miR-149*, hsa-miR-582-3p, hsa-miR-1227, hsa-miR-634, hsa-miR-576-5p and hsa-miR-641). These profiling results were validated by the detection of some selected miRNAs by qPCR. In silico analyses predicted that key molecular pathways potentially altered by the miRNAs differentially expressed in normal and OA chondrocytes include TGF-beta, Wnt, Erb and mTOR signalling; all of them implicated in the development, maintenance and destruction of articular cartilage. Conclusions. We have identified 7 miRNAs differentially expressed in OA and normal chondrocytes. Our potential miRNA target predictions and the signalling cascades altered by the differentially expressed miRNAs supports the potential involvement of the detected miRNAs in OA pathology. Due to the importance of miRNA in mediating the translation of target mRNA into protein, the identification of these miRNAs differentially expressed in normal and OA chondrocyte micropellets could have important diagnostic and therapeutic potential. Further studies are needed to know the function of these miRNAs, including the search of their target mRNA genes, which could lead to the development of novel therapeutic strategies for the OA treatment.Instituto de Salud Carlos III; CIBER BBN CB06-01-004

Mitochondrial Haplogroups Define Two Phenotypes of Osteoarthritis

Objective: To assess a mitochondrion-related phenotype in patients with osteoarthritis (OA). Methods: Serum levels of the following OA-related biomarkers: matrix metalloproteinase-1 (MMP-1); MMP-3; MMP-13; myeloperoxidase (MPO); a peptide of the alpha-helical region of type II collagen, Coll2-1, and its nitrated form Coll2-1NO2; a C-terminal neoepitope generated by the collagenase-mediated cleavage of collagen type II triple helix, C2C; the C-propeptide of collagen type II, CPII; hyaluronic acid (HA); human cartilage glycoprotein 39, YKL-40; cartilage oligomeric matrix protein; and cathepsin K were analyzed in 48 OA patients and 52 healthy controls carrying the haplogroups H and J. Logistic regression models and receiver operating characteristic (ROC) curves were performed to predict the onset of OA. Results: MMP-13 was the only biomarker significantly increased in OA patients compared to healthy controls in both haplogroups H and J. The collagen type II biomarkers, Coll2-1, Coll2-1NO2, the Coll2-1NO2/Coll2-1 ratio, C2C, CPII, and the C2C:CPII ratio were significantly increased in OA patients carrying haplogroup H compared to OA carriers of the haplogroup J. Two logistic regression models for diagnosis were constructed and adjusted for age, gender, and body mass index. For haplogroup H, the biomarkers significantly associated with OA were MMP-13 and Coll2-1; the area under the curve (AUC) of the ROC curve for this model was 0.952 (95% CI = 0.892–1.012). For haplogroup J, the only biomarker significantly associated with OA was MMP-13; the AUC for this model was 0.895 (95% CI = 0.801–0.989). Conclusion: The mitochondrial DNA haplogroups are potential complementary candidates for biomarkers of OA; their genotyping in conjunction with the assessment of classical protein molecular markers is recommended

Determinación del perfil proteico diferencial de líquido sinovial en pacientes con artrosis y artritis reumatoide mediante nanoLC acoplada a MALDI-TOF7TOF

Comunicaciones a congreso

Mitochondrial DNA (mtDNA) haplogroups and serum levels of anti-oxidant enzymes in patients with osteoarthritis

BACKGROUND: Oxidative stress play a main role in the initiation and progression of the OA disease and leads to the degeneration of mitochondria. To prevent this, the chondrocytes possess a well-coordinated enzymatic antioxidant system. Besides, the mitochondrial DNA (mtDNA) haplogroups are associated with the OA disease. Thus, the main goal of this work is to assess the incidence of the mtDNA haplogroups on serum levels of two of the main antioxidant enzymes, Manganese Superoxide Dismutase (Mn-SOD or SOD2) and catalase, and to test the suitability of these two proteins for potential OA-related biomarkers. METHODS: We analyzed the serum levels of SOD2 and catalase in 73 OA patients and 77 healthy controls carrying the haplogroups J, U and H, by ELISA assay. Knee and hip radiographs were classified according to Kellgren and Lawrence (K/L) scoring from Grade 0 to Grade IV. Appropriate statistical analyses were performed to test the effects of clinical variables, including gender, body mass index (BMI), age, smoking status, diagnosis, haplogroups and radiologic K/L grade on serum levels of these enzymes. RESULTS: Serum levels of SOD2 appeared statistically increased in OA patients when compared with healthy controls (p < 0.001). Even in those OA patients with higher OA severity (K/L grade IV), the serum levels of this antioxidant enzyme appeared more significantly increased than in OA patients with lower K/L grade (p < 0.001). The mtDNA haplogroups showed an influence on serum levels of catalase (p = 0.054), being carriers of the mtDNA haplogroup J those who showed higher serum levels than non-J carriers (p = 0.057). CONCLUSIONS: The increased levels of SOD2 in OA patients indicate an increased oxidative stress OA-related, therefore this antioxidant enzyme could be a suitable candidate biomarker for diagnosis of OA. Mitochondrial haplogroups significantly correlates with serum levels of catalase

Descripción de la cohorte PROCOAC (PROspective COhort of A Coruña): Cohorte prospectiva española para el estudio de la osteoartritis

[Abstract] Introduction and objective: The use of well characterized osteoarthritis (OA) cohorts is mandatory for the study and knowledge of this disease. Currently, there is no prospective cohort in this pathology in Spain. The objective of this work is to describe the first osteoarthritis cohort in Spain, PROCOAC (Cohort PROspectiva de A Coruña). Methods: The Unit of Rheumatology of the University Hospital of A Coruña started a prospective follow-up study in 2006. The patient inclusion criteria were: I) Patients older than 55 years who underwent an abdominal x-ray to study both hips II) Patients diagnosed with radiographic hand OA according to ACR criteria III) Patients diagnosed with radiographic knee or hip OA according to ACR criteria. Follow-up was performed every two years collecting clinical, analytical, genetic and radiographic information. Results: The cohort consists of 937 patients, 873 have radiographic knee OA, 783 hip OA and 679 hand OA. The mean age of the population is 63.9±8.9 years and the average BMI is 29.6±5.1. More than half of the population has high blood pressure and 17% diabetes. The predominant osteoarthritis in the hand is nodular (78.1%), followed by trapeziometacarpal (55.3%) and erosive (18.4%). Twenty-one point four percent and 43.1% are healthy at knee and hip level respectively; observing a grade 1 in 26% and 37%; a grade 2 in 26.7% and 11.5%; a grade 3 in 14.9% and 4%; and a grade 4 in 9.4% and 3.7% respectively. Of the population, 44.1% has only 1 joint affected, 39.9% has 2 and 13.4% has 3 joints affected. Age (OR=1.11; p<.001), BMI (OR=1.11; p=.002) and total WOMAC (OR=1.03; p=.005) are the only risk factors if we compare the involvement of a single location versus three. A discrepancy between pain and radiographic damage at the joint level was also detected in patients with KL≤2 grade, and therefore a significantly higher percentage of patients with knee OA experienced pain (66.1%) compared to patients with OA hip (21.1%) (p<.001). Conclusions: The PROCOAC cohort is an instrument that allows studies of incidence and progression in hand, knee and hip OA; as well as determining factors that are associated with the different OA phenotypes.[Resumen] Introducción y objetivo. El uso de cohortes de Osteoartritis (OA) bien caracterizadas es obligatorio para estudiar y profundizar en el conocimiento en esta enfermedad. En España no existe actualmente ninguna cohorte prospectiva en esta patología; así el objetivo de este trabajo es describir la primera cohorte de Osteoartritis en España, la PROCOAC (PROspective COhort of A Coruña). Material y métodos. El Servicio de Reumatología del Hospital Universitario de A Coruña inició un estudio de seguimiento prospectivo en el año 2006. Los criterios de inclusión fueron: I) Pacientes mayores de 55 años a los que se les realizó una radiografía abdominal que permitiese estudiar ambas caderas II) Pacientes diagnosticados de OA radiográfica de mano según los criterios ACR III) Pacientes diagnosticados de OA radiográfica de rodilla y/o cadera según los criterios ACR. Se realizó seguimiento cada dos años recogiendo información clínica, analítica, genética y radiográfica. Resultados. La cohorte consta de 937 individuos, 873 tienen OA radiográfica de rodilla, 783 de cadera y 679 de mano. La edad media de la población es 63,9 ± 8,9 años y el IMC promedio de 29,6 ± 5,1. Más de la mitad de la población tiene hipertensión arterial y el 17% diabetes. La osteoartritis predominate en la mano es la nodular (78,1%), seguida de la rizartrosis (55,3%) y la erosiva (18,4%). El 21,4% y el 43,1% son sanos a nivel de rodilla y cadera respectivamente; observando un grado 1 en el 26% y 37%; un grado 2 en el 26,7% y 11,5%; un grado 3 en el 14,9% y 4%; y un grado 4 en el 9,4% y 3,7% respectivamente. El 44,1% de la población tiene 1 articulación afectada, el 39,9% tiene 2 y el 13,4% tiene 3 articulaciones afectadas. La edad (OR = 1,11; p < 0,001), el IMC (OR = 1,11; p = 0,002) y el WOMAC total (OR = 1,03; p = 0,005) son los únicos factores de riesgo si comparamos la afectación de una sola ubicación frente a tres. También se detectó una discrepancia entre el dolor y el daño radiográfico a nivel articular en pacientes con grado KL ≤ 2, de modo que un porcentaje significativamente mayor de pacientes con OA de rodilla experimentaron dolor (66,1%) en comparación con pacientes con OA de cadera (21,1%) (p < 0,001). Conclusión. La cohorte PROCOAC es un instrumento que permite realizar estudios de incidencia y pro-gresión en la osteoartritis de mano, rodilla y cadera, así como conocer factores que se asocian con losdiferentes fenotipos de osteoartritis

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

Defective chaperone-mediated autophagy is a hallmark of joint disease in patients with knee osteoarthritis

[Abstract] Objective: Defects in autophagy contribute to joint aging and Osteoarthritis (OA). Identifying specific autophagy types could be useful for developing novel treatments for OA. Design: An autophagy-related gene array was performed in blood from non-OA and knee OA subjects from the Prospective Cohort of A Coruña (PROCOAC). The differential expression of candidate genes was confirmed in blood and knee cartilage and a regression analysis was performed adjusting for age and BMI. HSP90A, a chaperone mediated autophagy (CMA) marker was validated in human knee joint tissues, as well as, in mice with aging-related and surgically-induced OA. The consequences of HSP90AA1 deficiency were evaluated on OA pathogenesis. Finally, the contribution of CMA to homeostasis was studied by assessing the capacity to restore proteostasis upon ATG5-mediated macroautophagy deficiency and genetic HSP90AA1 overexpression. Results: 16 autophagy-related genes were significantly down-regulated in blood from knee OA subjects. Validation studies showed that HSP90AA1 was down-regulated in blood and human OA cartilage and correlated with risk incidence of OA. Moreover, HSP90A was reduced in human OA joints tissues and with aging and OA in mice. HSP90AA1 knockdown was linked to defective macroautophagy, inflammation, oxidative stress, senescence and apoptosis. However, macroautophagy deficiency increased CMA, highlighting the CMA-macroautophagy crosstalk. Remarkably, CMA activation was sufficient to protect chondrocytes from damage. Conclusions: We show that HSP90A is a key chaperone for chondrocyte homeostasis, while defective CMA contributes to joint damage. We propose that CMA deficiency is a relevant disease mechanism and could represent a therapeutic target for OA.Instituto de Salud Carlos III; PI17/02059Instituto de Salud Carlos III; PI20/0064