Higher Synovial Immunohistochemistry Reactivity of IL-17A, Dkk1, and TGF-ß1 in Patients with Early Psoriatic Arthritis and Rheumatoid Arthritis Could Predict the Use of Biologics.

BACKGROUND: Delay in diagnosis and therapy in patients with arthritis commonly leads to progressive articular damage. The study aimed to investigate the immunohistochemical reactivity of synovial cytokines associated with inflammation and the bone erosives/neoformatives processes among individuals diagnosed with psoriatic arthritis (PsA), rheumatoid arthritis (RA), osteoarthritis (OA), and radiographic axial spondyloarthritis (r-axSpA), with the intention of identifying potential biomarkers. METHODS: Specimens were collected from the inflamed knee joints of patients referred for arthroscopic procedures, and the synovial tissue (ST) was prepared for quantifying protein expression through immunohistochemical analysis (% expressed in Ratio_Area-Intensity) for TGF-ß1, IL-17A, Dkk1, BMP2, BMP4, and Wnt5b. The collected data underwent thorough analysis and examination of their predictive capabilities utilising receiver operating characteristic (ROC) curves. RESULTS: Valid synovial tissue samples were acquired from 40 patients for IHC quantification analysis. Initially, these patients had not undergone treatment with biologics. However, after 5 years, 4 out of 13 patients diagnosed with PsA and two out of nine patients diagnosed with RA had commenced biologic treatments. Individuals with early PsA who received subsequent biologic treatment exhibited significantly elevated IHC reactivity in ST for TGF-ß1 (p = 0.015). Additionally, patients with both PsA and RA who underwent biologic therapy displayed increased IHC reactivity for IL-17A (p = 0.016), TGF-ß1 (p = 0.009), and Dkk1 (p = 0.042). ROC curve analysis of IHC reactivity for TGF-ß1, Dkk1, and IL-17A in the synovial seems to predict future treatment with biologics in the next 5 years with the area under the curve (AUC) of a combined sum of the three values: AUC: 0.828 (95% CI: 0.689-0.968; p 0.005) S 75% E 84.4%. CONCLUSIONS: Higher synovial immunohistochemistry reactivity of IL-17A, Dkk1, and TGF-ß1 in patients with early psoriatic arthritis and rheumatoid arthritis may serve as potential indicators for predicting the necessity of utilising biologic treatments.

Mitonuclear epistasis involving TP63 and haplogroup Uk: Risk of rapid progression of knee OA in patients from the OAI.

OBJECTIVE: To investigate genetic interactions between mitochondrial deoxyribonucleic acid (mtDNA) haplogroups and nuclear single nucleotide polymorphisms (nSNPs) to analyze their impact on the development of the rapid progression of knee osteoarthritis (OA). DESIGN: A total of 1095 subjects from the Osteoarthritis Initiative, with a follow-up time of at least 48-months, were included. Appropriate statistical approaches were performed, including generalized estimating equations adjusting for age, gender, body mass index, contralateral knee OA, Western Ontario and McMaster Universities Osteoarthritis Index pain, previous injury in target knee and the presence of the mtDNA variant m.16519C. Additional genomic data consisted in the genotyping of Caucasian mtDNA haplogroups and eight nSNPs previously associated with the risk of knee OA in robust genome-wide association studies. RESULTS: The simultaneous presence of the G allele of rs12107036 at TP63 and the haplogroup Uk significantly increases the risk of a rapid progression of knee OA (odds ratio = 1.670; 95% confidence interval [CI]: 1.031-2.706; adjusted p-value = 0.027). The assessment of the population attributable fraction showed that the highest proportion of rapid progressors was under the simultaneous presence of the G allele of rs12107036 and the haplogroup Uk (23.4%) (95%CI: 7.89-38.9; p-value < 0.05). The area under the curve of the cross-validation model (0.730) was very similar to the obtained for the predictive model (0.735). A nomogram was constructed to help clinicians to perform clinical trials or epidemiologic studies. CONCLUSIONS: This study demonstrates the existence of a mitonuclear epistasis in OA, providing new mechanisms by which nuclear and mitochondrial variation influence the susceptibility to develop different OA phenotypes.

Is osteoarthritis a mitochondrial disease? What is the evidence.

PROPOSE OF REVIEW: To summarize the evidence that suggests that osteoarthritis (OA) is a mitochondrial disease. RECENT FINDINGS: Mitochondrial dysfunction together with mtDNA damage could contribute to cartilage degradation via several processes such as: (1) increased apoptosis; (2) decreased autophagy; (3) enhanced inflammatory response; (4) telomere shortening and increased senescence chondrocytes; (5) decreased mitochondrial biogenesis and mitophagy; (6) increased cartilage catabolism; (7) increased mitochondrial fusion leading to further reactive oxygen species production; and (8) impaired metabolic flexibility. SUMMARY: Mitochondria play an important role in some events involved in the pathogenesis of OA, such as energy production, the generation of reactive oxygen and nitrogen species, apoptosis, authophagy, senescence and inflammation. The regulation of these processes in the cartilage is at least partially controlled by retrograde regulation from mitochondria and mitochondrial genetic variation. Retrograde regulation through mitochondrial haplogroups exerts a signaling control over the nuclear epigenome, which leads to the modulation of nuclear genes, cellular functions and development of OA. All these data suggest that OA could be considered a mitochondrial disease as well as other complex chronic disease as cancer, cardiovascular and neurologic diseases.

Oleate Prevents Palmitate-Induced Mitochondrial Dysfunction in Chondrocytes.

The association between obesity and osteoarthritis (OA) in joints not subjected to mechanical overload, together with the relationship between OA and metabolic syndrome, suggests that there are systemic factors related to metabolic disorders that are involved in the metabolic phenotype of OA. The aim of this work is study the effects of palmitate and oleate on cellular metabolism in an "in vitro" model of human chondrocytes. The TC28a2 chondrocyte cell line was used to analyze the effect of palmitate and oleate on mitochondrial and glycolytic function, Adenosine triphosphate (ATP) production and lipid droplets accumulation. Palmitate, but not oleate, produces mitochondrial dysfunction observed with a lower coupling efficiency, maximal respiration and spare respiratory capacity. Glycolytic function showed lower rates both glycolytic capacity and glycolytic reserve when cells were incubated with fatty acids (FAs). The production rate of total and mitochondrial ATP showed lower values in chondrocytes incubated with palmitic acid (PA). The formation of lipid droplets increased in FA conditions, being significantly higher when the cells were incubated with oleic acid (OL). These results may help explain, at least in part, the close relationship of metabolic pathologies with OA, as well as help to elucidate some of the factors that can define a metabolic phenotype in OA.

Design of a digital-PCR assay to quantify fragmented human mitochondrial DNA.

Digital PCR (dPCR) has been adapted to quantify the proportion of mitochondrial DNA (mtDNA) molecules without and with double-strand DNA breaks (DSBs). This is based on a break-apart approach of two differentially labeled target sequences distantly located in the circular DNA molecule. When the two targets amplify in separated reaction partitions, the original mtDNA molecule should be fragmented by two DSBs at least, each in a different segment between targets. When both targets amplify in the same partition, it must correspond to a circular or linear mtDNA molecule. These two possibilities may be distinguished through a restriction endonuclease (RE) induced unique DSB within a DNA segment between the targets. After RE-digestion, separation of both target signals in different partitions must indicate the presence of a previous linear mtDNA molecule. Otherwise, joint amplification in the same partition would correspond to an initial circular mtDNA that has been linearized by the endonuclease. The procedure was validated by assaying different proportions of mtDNA fragmented by in vitro digestion with REs, evidencing a perfect accordance between the expected theoretical values and dPCR quantification. Samples from peripheral blood cells, cellular and extracellular DNA from the U2OS cell line, as well as cells incubated with ethidium bromide to induce mtDNA depletion, were evaluated. The technique may be of interest to complement the studies of mtDNA in relation to aging and human disease, as well as to assess possible adverse effects of certain drugs that could be related to affectation of mtDNA.

Discovery of an autoantibody signature for the early diagnosis of knee osteoarthritis: data from the Osteoarthritis Initiative.

OBJECTIVE: To find autoantibodies (AAbs) in serum that could be useful to predict incidence of radiographic knee osteoarthritis (KOA). DESIGN: A Nucleic-acid Programmable Protein Arrays (NAPPA) platform was used to screen AAbs against 2125 human proteins in sera at baseline from participants free of radiographic KOA belonging to the incidence and non-exposed subcohorts of the Osteoarthritis Initiative (OAI) who developed or not, radiographic KOA during a follow-up period of 96 months. NAPPA-ELISA were performed to analyse reactivity against methionine adenosyltransferase two beta (MAT2ß) and verify the results in 327 participants from the same subcohorts. The association of MAT2ß-AAb levels with KOA incidence was assessed by combining several robust biostatistics analysis (logistic regression, Receiver Operating Characteristic and Kaplan-Meier curves). The proposed prognostic model was replicated in samples from the progression subcohort of the OAI. RESULTS: In the screening phase, six AAbs were found significantly different at baseline in samples from incident compared with non-incident participants. In the verification phase, high levels of MAT2ß-AAb were significantly associated with the future incidence of KOA and with an earlier development of the disease. The incorporation of this AAb in a clinical model for the prognosis of incident radiographic KOA significantly improved the identification/classification of patients who will develop the disorder. The usefulness of the model to predict radiographic KOA was confirmed on a different OAI subcohort. CONCLUSIONS: The measurement of AAbs against MAT2ß in serum might be highly useful to improve the prediction of OA development, and also to estimate the time to incidence.

Mitochondrial DNA haplogroups influence the risk of aortic stenosis.

AIM: The underlying pathophysiologic mechanisms of aortic stenosis are not clear. Mitochondrial dysfunction plays a role in many pathological conditions including cardiac diseases. We aimed to analyze the mitochondrial DNA haplogroups in a group of patients undergoing valve replacement surgery due to severe aortic stenosis. METHODS: Mitochondrial DNA haplogroups were assessed in 176 patients with severe aortic stenosis and 308 control subjects. Cardiovascular risk factors and demographics were similar in both groups. RESULTS: Patients carrying haplogroup Uk had a lower risk of developing aortic stenosis, especially compared to patients carrying haplogroup H (odds ratio = 0.507; 95% confidence interval: 0.270-0.952, p = 0.035). CONCLUSIONS: Mitochondrial DNA haplogroups could be involved in the development of severe aortic stenosis. Specifically, haplogroup H could be a risk factor and Uk a protective factor for severe aortic stenosis in a population from Spain.

Generating Rho-0 Cells Using Mesenchymal Stem Cell Lines.

INTRODUCTION: The generation of Rho-0 cells requires the use of an immortalization process, or tumor cell selection, followed by culture in the presence of ethidium bromide (EtBr), incurring the drawbacks its use entails. The purpose of this work was to generate Rho-0 cells using human mesenchymal stem cells (hMSCs) with reagents having the ability to remove mitochondrial DNA (mtDNA) more safely than by using EtBr. METHODOLOGY: Two immortalized hMSC lines (3a6 and KP) were used; 143B.TK-Rho-0 cells were used as reference control. For generation of Rho-0 hMSCs, cells were cultured in medium supplemented with each tested reagent. Total DNA was isolated and mtDNA content was measured by real-time polymerase chain reaction (PCR). Phenotypic characterization and gene expression assays were performed to determine whether 3a6 Rho-0 hMSCs maintain the same stem properties as untreated 3a6 hMSCs. To evaluate whether 3a6 Rho-0 hMSCs had a phenotype similar to that of 143B.TK-Rho-0 cells, in terms of reactive oxygen species (ROS) production, apoptotic levels and mitochondrial membrane potential (Δψm) were measured by flow cytometry and mitochondrial respiration was evaluated using a SeaHorse XFp Extracellular Flux Analyzer. The differentiation capacity of 3a6 and 3a6 Rho-0 hMSCs was evaluated using real-time PCR, comparing the relative expression of genes involved in osteogenesis, adipogenesis and chondrogenesis. RESULTS: The results showed the capacity of the 3a6 cell line to deplete its mtDNA and to survive in culture with uridine. Of all tested drugs, Stavudine (dt4) was the most effective in producing 3a6-Rho cells. The data indicate that hMSC Rho-0 cells continue to express the characteristic MSC cell surface receptor pattern. Phenotypic characterization showed that 3a6 Rho-0 cells resembled 143B.TK-Rho-0 cells, indicating that hMSC Rho-0 cells are Rho-0 cells. While the adipogenic capability was higher in 3a6 Rho-0 cells than in 3a6 cells, the osteogenic and chondrogenic capacities were lower. CONCLUSION: Among the drugs and conditions tested, the use of d4t was the best option for producing Rho-0 cells from hMSCs. Rho-0 cells are useful for studying the role of mitochondria in hMSC differentiation.

Mitochondrial DNA haplogroups modulate the radiographic progression of Spanish patients with osteoarthritis.

Not all patients with osteoarthritis (OA) show the same disease progression, as some of them remain relatively stable over time, while others progress to severe structural deterioration of the joint. In this sense, the main goal of both genetic and protein biomarkers in OA is to predict not only the risk of OA at an earlier stage of the disease but also which OA patients are more likely to progress to severe disease. Taking into account the incidence of the mitochondria and the mtDNA haplogroups in the pathogenesis of OA, the main objective of this work was to evaluate the incidence of the mtDNA haplogroups in the radiographic progression of the OA disease in a well-characterized follow-up cohort of Spanish patients. DNA from 281 OA patients from Hospital Universitario A Coruña was isolated to determine the European mtDNA haplogroups. Knee or hip radiographs from all affected joints were obtained at two time points with at least 36 months apart. Radiographs were evaluated using the Kellgren/Lawrence (K/L) scale; radiographic OA progression was defined as any radiographic worsening of the K/L joint score. Statistical analyses included Kaplan-Meier survival curves and Cox regression models. Patients belonging to the cluster TJ showed a slower radiographic OA progression than patients in the cluster KU (p = 0.036). Moreover, patients carrying the most common mtDNA haplogroup H are more apt to require total joint replacement surgery than non-H patients (p = 0.049). The inherited mitochondrial variants influence the radiographic progression of OA and could be considered among the genetic variants taken into account when the radiographic progression of OA is analyzed.

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.