Mitochondrial Role on Cellular Apoptosis, Autophagy, and Senescence during Osteoarthritis Pathogenesis.

Authors have demonstrated that apoptosis activation is a pathway related to cartilage degradation characteristics of the OA process. Autophagy is an adaptive response to protect cells from various environmental changes, and defects in autophagy are linked to cell death. In this sense, decreased autophagy of chondrocytes has been observed in OA articular cartilage. The aim of this work was to study the role of OA mitochondria in apoptosis, autophagy, and senescence, using OA and Normal (N) transmitochondrial cybrids. Results: OA cybrids incubated with menadione showed a higher percentage of late apoptosis and necrosis than N cybrids. Stimulation of cybrids with staurosporine and IL-1ß showed that OA cybrids were more susceptible to undergoing apoptosis than N cybrids. An analysis of the antioxidant response using menadione on gene expression revealed a lower expression of nuclear factor erythroid 2-like 2 and superoxide dismutase 2 in OA than N cybrids. Activation of microtubule-associated protein 1A/1B-light chain 3 was reduced in OA compared to N cybrids. However, the percentage of senescent cells was higher in OA than N cybrids. Conclusion: This work suggests that mitochondria from OA patients could be involved in the apoptosis, autophagy, and senescence of chondrocytes described in OA cartilage.

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.

Anti-Inflammatory Activity of APPA (Apocynin and Paeonol) in Human Articular Chondrocytes.

Osteoarthritis (OA) is a chronic joint disease leading to cartilage loss and reduction in the joint space which results in pain. The current pharmacological treatment of OA is inadequate and pharmacological interventions focus on symptom management. APPA, a combination of apocynin (AP) and paeonol (PA), is a potential drug for treating OA. The aim of this study was to analyze the effects of APPA on the modulation of the inflammatory response in chondrocytes. Samples were incubated with IL-1ß and APPA, and their responses to proinflammatory cytokines, catabolic mediators and redox responses were then measured. The effect of APPA on mitogenesis was also evaluated. Results show that APPA attenuated the expression of IL-8, TNF-α, MMP-3, MMP-13, SOD-2 and iNOS, resulting in the protection of human articular cartilage. APPA decreased PGC-1α gene expression induced by IL-1ß. APPA did not modulate the gene expression of Mfn2, Sirt-1 or Sirt-3. The overall findings indicate that APPA may be an effective treatment for OA by targeting several of the pathways involved in OA pathogenesis.

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.

A meta-analysis and a functional study support the influence of mtDNA variant m.16519C on the risk of rapid progression of knee osteoarthritis.

OBJECTIVES: To identify mitochondrial DNA (mtDNA) genetic variants associated with the risk of rapid progression of knee osteoarthritis (OA) and to characterise their functional significance using a cellular model of transmitochondrial cybrids. METHODS: Three prospective cohorts contributed participants. The osteoarthritis initiative (OAI) included 1095 subjects, the Cohort Hip and Cohort Knee included 373 and 326 came from the PROspective Cohort of Osteoarthritis from A Coruña. mtDNA variants were screened in an initial subset of 450 subjects from the OAI by in-depth sequencing of mtDNA. A meta-analysis of the three cohorts was performed. A model of cybrids was constructed to study the functional consequences of harbouring the risk mtDNA variant by assessing: mtDNA copy number, mitochondrial biosynthesis, mitochondrial fission and fusion, mitochondrial reactive oxygen species (ROS), oxidative stress, autophagy and a whole transcriptome analysis by RNA-sequencing. RESULTS: mtDNA variant m.16519C is over-represented in rapid progressors (combined OR 1.546; 95% CI 1.163 to 2.054; p=0.0027). Cybrids with this variant show increased mtDNA copy number and decreased mitochondrial biosynthesis; they produce higher amounts of mitochondrial ROS, are less resistant to oxidative stress, show a lower expression of the mitochondrial fission-related gene fission mitochondrial 1 and an impairment of autophagic flux. In addition, its presence modulates the transcriptome of cybrids, especially in terms of inflammation, where interleukin 6 emerges as one of the most differentially expressed genes. CONCLUSIONS: The presence of the mtDNA variant m.16519C increases the risk of rapid progression of knee OA. Among the most modulated biological processes associated with this variant, inflammation and negative regulation of cellular process stand out. The design of therapies based on the maintenance of mitochondrial function is recommended.

mtDNA haplogroup A enhances the effect of obesity on the risk of knee OA in a Mexican population.

To evaluate the influence of mitochondrial DNA haplogroups on the risk of knee OA in terms of their interaction with obesity, in a population from Mexico. Samples were obtained from (n = 353) knee OA patients (KL grade ≥ I) and (n = 364) healthy controls (KL grade = 0) from Mexico city and Torreon (Mexico). Both Caucasian and Amerindian mtDNA haplogroups were assigned by single base extension assay. A set of clinical and demographic variables, including obesity status, were considered to perform appropriate statistical approaches, including chi-square contingency tables, regression models and interaction analyses. To ensure the robustness of the predictive model, a statistical cross-validation strategy of B = 1000 iterations was used. All the analyses were performed using boot, GmAMisc and epiR package from R software v4.0.2 and SPSS software v24. The frequency distribution of the mtDNA haplogroups between OA patients and healthy controls for obese and non-obese groups showed the haplogroup A as significantly over-represented in knee OA patients within the obese group (OR 2.23; 95% CI 1.22-4.05; p-value = 0.008). The subsequent logistic regression analysis, including as covariate the interaction between obesity and mtDNA haplogroup A, supported the significant association of this interaction (OR 2.57; 95% CI 1.24-5.32; p-value = 0.011). The statistical cross-validation strategy confirmed the robustness of the regression model. The data presented here indicate a link between obesity in knee OA patients and mtDNA haplogroup A.

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.

Soluble inflammatory mediators of synoviocytes stimulated by monosodium urate crystals induce the production of oxidative stress, pain, and inflammation mediators in chondrocytes : Secretome of synoviocytes induces chondrocyte damage.

We hypothesized that the secretion of inflammatory mediators from synoviocytes affects the chondrocyte homeostasis of articular cartilage. This study was a preliminary attempt to elucidate the molecular mechanisms by which soluble mediators obtained from activated synoviocytes induce oxidative stress and inflammation in chondrocytes. We measured the concentrations of interleukin-6 (IL-6), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), nerve growth factor (NGF), superoxide anion (O2•-), hydrogen peroxide (H2O2), and nitric oxide (NO•) from articular human cells. First, we created a conditional basal medium by exposing synoviocytes (HS) to monosodium urate crystals (CBM). The chondrocytes were exposed to either CBM (CCM), urate crystals directly (CMSU), or remained untreated (CC) as a negative control. Data were analyzed by ANOVA tests; Bonferroni test was performed for multiple comparisons between groups. Interestingly, we observed that mediators of inflammation and oxidative stress were significantly higher in CCM than CMSU and CC groups (P<0.01). The specific concentrations were as follows: 19.85 ng/mL of IL-6, 9.79 ng/mL of IL-8, 5.17 ng/mL of NGF, and 11.91 ng/mL of MCP-1. Of note, we observed the same trend for reactive oxygen and nitrogen species (P<0.001). Soluble mediators secreted by synoviocytes after being activated with MSU crystals (as observed in individuals who present gout attacks) trigger chondrocyte activation intensifying the articular inflammatory, oxidative, and pain states that damage cartilage in OA; this damage is more severe even when compared to HC directly exposed to monosodium urate crystals. Key Points • The molecular relation between MSU depositions and cartilage damage could be mediated by pro-inflammatory soluble mediators and oxidative molecules. • The secretion of pro-inflammatory mediators by activated synoviocytes is more harmful to chondrocytes than a direct activation in the chondrocyte culture. • Under this model, there is an important imbalance in the matrix homeostasis due to changes in several chemokines, cytokines, and other factors such as NGF, as well as oxidative mediators.

Mitochondrial DNA from osteoarthritic patients drives functional impairment of mitochondrial activity: a study on transmitochondrial cybrids.

With the redefinition of osteoarthritis (OA) and the understanding that the joint behaves as an organ, OA is now considered a systemic illness with a low grade of chronic inflammation. Mitochondrial dysfunction is well documented in OA and has the capacity to alter chondrocyte and synoviocyte function. Transmitochondrial cybrids are suggested as a useful cellular model to study mitochondrial biology in vitro, as they carry different mitochondrial variants with the same nuclear background. The aim of this work was to study mitochondrial and metabolic function of cybrids with mitochondrial DNA from healthy (N) and OA donors. In this work, the authors demonstrate that cybrids from OA patients behave differently from cybrids from N donors in several mitochondrial parameters. Furthermore, OA cybrids behave similarly to OA chondrocytes. These results enhance our understanding of the role of mitochondria in the degeneration process of OA and present cybrids as a useful model to study OA pathogenesis.

Postoperative Plasma Mitochondrial DNA and Cytokine Profiles of Elderly Patients Undergoing Minimally Invasive Aortic Valve Replacement.

INTRODUCTION: Mitochondrial DNA (mtDNA) is gaining increasing interest as a marker of cellular damage and could also act as an inflammatory mediator in cardiopulmonary bypass induced postoperative inflammatory response. Although minimally invasive heart valve surgery reportedly reduces inflammation, the mtDNA and cytokine profile in this context remains unclear. MATERIALS AND METHODS: Here, we report a prospective series of 40 elderly patients with aortic stenosis who underwent bioprosthetic aortic valve replacement (AVR) through upper ministernotomy with either a sutureless (n = 20) or a conventional (n = 20) valve. Primary end points included serial plasma levels of mtDNA (T1: at baseline; T2: 4 hours after surgery; and T3: 24s hour after surgery), cytokines (interleukin-6 [IL-6], tumor necrosis factor-α [TNF-α]), and myocardial necrosis biomarkers (MNBs), whereas secondary end points included clinical and echocardiographic data. RESULTS: Significant increases in the postoperative plasma levels (T2) of mtDNA, cytokines, and MNBs were observed in all patients. The postoperative plasma levels of mtDNA, TNF-α, and MNBs showed no significant differences between the treatment groups, although there was a trend toward lower levels in the sutureless group. The decreases in aortic cross-clamp and cardiopulmonary bypass times seen in the sutureless group were associated with significant lower postoperative levels (T2 and T3) of IL-6. CONCLUSION: AVR through upper ministernotomy was associated with a significant increase in postoperative plasma levels of mtDNA and cytokines. There was no difference in the mtDNA levels between the sutureless and conventional valve groups, suggesting a similar level of inflammation in both groups. However, the shorter operation time observed in the sutureless valve group was associated with significantly lower postoperative levels of IL-6, indicating potential clinical benefits.

Impaired Metabolic Flexibility in the Osteoarthritis Process: A Study on Transmitochondrial Cybrids.

Osteoarthritis (OA) is the most frequent joint disease; however, the etiopathogenesis is still unclear. Chondrocytes rely primarily on glycolysis to meet cellular energy demand, but studies implicate impaired mitochondrial function in OA pathogenesis. The relationship between mitochondrial dysfunction and OA has been established. The aim of the study was to examine the differences in glucose and Fatty Acids (FA) metabolism, especially with regards to metabolic flexibility, in cybrids from healthy (N) or OA donors. Glucose and FA metabolism were studied using D-[14C(U)]glucose and [1-14C]oleic acid, respectively. There were no differences in glucose metabolism among the cybrids. Osteoarthritis cybrids had lower acid-soluble metabolites, reflecting incomplete FA ß-oxidation but higher incorporation of oleic acid into triacylglycerol. Co-incubation with glucose and oleic acid showed that N but not OA cybrids increased their glucose metabolism. When treating with the mitochondrial inhibitor etomoxir, N cybrids still maintained higher glucose oxidation. Furthermore, OA cybrids had higher oxidative stress response. Combined, this indicated that N cybrids had higher metabolic flexibility than OA cybrids. Healthy donors maintained the glycolytic phenotype, whereas OA donors showed a preference towards oleic acid metabolism. Interestingly, the results indicated that cybrids from OA patients had mitochondrial impairments and reduced metabolic flexibility compared to N cybrids.

Wnt and RUNX2 mediate cartilage breakdown by osteoarthritis synovial fibroblast-derived ADAMTS-7 and -12.

Failure of therapeutic approaches for the treatment of osteoarthritis (OA) based on the inhibition of metalloproteinases, might be because of their constitutive expression in homeostasis, together with their network complexity. The knowledge of this network would contribute to selective target pathological conditions. In this sense, blockade of mediators produced by neighbouring joint cells, such as synovial fibroblasts (SF), would prevent cartilage damage. Thus, we studied the contribution of ADAMTS-7 and -12 from SF to cartilage oligomeric matrix protein (COMP) degradation, and the signalling pathways involved in their expression. We report for the first time in SF, the involvement of ERK-Runx2 axis and Wnt/ß-catenin signalling in ADAMTS-12 and ADAMTS-7 expressions, respectively, with the subsequent consequences in COMP degradation from cartilage extracellular matrix. After stimulation with IL-1ß or fibronectin fragments, we showed that ERK inhibition decreased Runx2 activation and ADAMTS-12 expression in OA-SF, also reducing Fn-fs-induced COMP degradation. Blockage of Wnt signalling by DKK1 reduced ADAMTS-7 and COMP degradation in OA-SF as well. In addition, Wnt7B expression was induced by IL-1ß and by itself, also increasing ADAMTS-7. Our results could contribute to the development of disease-modifying OA drugs targeting ADAMTS-7 and -12 for the prevention of extracellular matrix components degradation like COMP.

Differential Association of Mitochondrial DNA Haplogroups J and H With the Methylation Status of Articular Cartilage: Potential Role in Apoptosis and Metabolic and Developmental Processes.

OBJECTIVE: To analyze the influence of mitochondrial genome variation on the DNA methylome of articular cartilage. METHODS: DNA methylation profiling was performed using data deposited in the NCBI Gene Expression Omnibus database (accession no. GSE43269). Data were obtained for 14 cartilage samples from subjects with haplogroup J and 20 cartilage samples from subjects with haplogroup H. Subsequent validation was performed in an independent subset of 7 subjects with haplogroup J and 9 with haplogroup H by RNA-seq. Correlated genes were validated by real-time polymerase chain reaction in an independent cohort of 12 subjects with haplogroup J and 12 with haplogroup H. Appropriate analyses were performed using R Bioconductor and qBasePlus software, and gene ontology analysis was conducted using DAVID version 6.8. RESULTS: DNA methylation profiling revealed 538 differentially methylated loci, while whole-transcriptome profiling identified 2,384 differentially expressed genes, between cartilage samples from subjects with haplogroup H and those with haplogroup J. Seventeen genes showed an inverse correlation between methylation and expression. In terms of gene ontology, differences in correlations between methylation and expression were also detected between cartilage from subjects with haplogroup H and those with haplogroup J, highlighting a significantly enhanced apoptotic process in cartilage from subjects with haplogroup H (P = 0.007 for methylation and P = 0.019 for expression) and repressed apoptotic process in cartilage from subjects with haplogroup J (P = 0.021 for methylation), as well as a significant enrichment of genes related to metabolic processes (P = 1.93 × 10-4 for methylation and P = 6.79 x 10-4 for expression) and regulation of gene expression (P = 0.012 for methylation) in cartilage from subjects with haplogroup H, and to developmental processes (P = 0.015 for methylation and P = 8.25 x 10-12 for expression) in cartilage from subjects with haplogroup J. CONCLUSION: Mitochondrial DNA variation differentially associates with the methylation status of articular cartilage by acting on key mechanisms involved in osteoarthritis, such as apoptosis and metabolic and developmental processes.

In vitro comprehensive analysis of VA692 a new chemical entity for the treatment of osteoarthritis.

Selective cyclooxigenase (COX)-2 inhibitors were developed to prevent traditional non-steroidal anti-inflammatory drugs (tNSAIDs) gastro-intestinal adverse effects. VA692, a recently disclosed selective COX-2 inhibitor, structurally related to well-known marketed coxibs, showed anti-inflammatory, and anti-nociceptive properties. The aim of this study was to analyze the anti-inflammatory effect of VA692, in comparison with celecoxib. At this purpose we evaluated the pro-inflammatory cytokines and anti-oxidant enzymes gene expression, apoptosis and ROS production, and PGE2 release in chondrocytes (both primary cultures and immortalized T/C-28a2 cell line) treated with the two drugs. Furthermore, a proteomic analysis has been performed in T/C-28a2 cell line to evaluate modifications in their proteomic profile following drug treatment in presence of IL-1ß. Our results demonstrated the anti-inflammatory effect of the novel synthesized VA692, and confirmed those of celecoxib, in counteracting the stimulus of IL-1ß in both osteoarthritic (OA) chondrocytes and T/C-28a2 cell line. Furthermore, the data underlined the possible anti-apoptotic and anti-oxidant role of VA692, implying its regulation in superoxide anion production as indicated by the modulation of anti-oxidant enzymes. The proteomic analysis provides new information about the effect of VA692 on human T/C-28a2 intracellular proteome, demonstrating the usefulness of this approach in the identification and quantifications of several proteins. Modulation of some proteins such as Hsp90 and SOD by VA692 could explain its role in the therapeutic approach of OA. Based on our results, we can affirm that VA692 has more beneficial effect compared with celecoxib particularly regarding the modulation of oxidant/anti-oxidant system and proteome profile of human articular chondrocytes.

A replication study and meta-analysis of mitochondrial DNA variants in the radiographic progression of knee osteoarthritis.

OBJECTIVE: To conduct a replication study and meta-analysis involving the study of mtDNA variants in the radiographic progression of OA in different cohorts worldwide, including Cohort Hip and Cohort Knee (CHECK), the OA Initiative and a cohort from Spain. METHODS: The influence of the haplogroups in the rate of radiographic progression at 96 months in 431 subjects from CHECK was assessed in terms of Kellgren and Lawrence (KL) grade. Progression was defined as a change from KL ⩾ 1 at baseline to any higher grade during the follow-up. Extended Cox proportional hazard models were used to analyse the influence of mtDNA variants in the rate of radiographic knee OA progression. A subsequent meta-analysis of 1603 subjects following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines was conducted to combine the data of individual studies. A sensitivity analysis was performed to validate the stability of the results. RESULTS: CHECK subjects carrying the haplogroup T showed the lowest rate of radiographic knee OA progression [hazard ratio (HR) 0.645 (95% CI 0.419, 0.978); P < 0.05]. When pooled, subjects within the superhaplogroup JT showed the same trend [HR 0.707 (95% CI 0.501, 0.965); P < 0.05]. BMI [HR 1.046 (95% CI 1.018, 1.073); P < 0.05] and bilateral OA [HR 2.266 (95% CI 1.733, 2.954); P < 0.05] at baseline are risk factors for radiographic knee OA progression as well. In the meta-analysis there was a reduced rate of radiographic progression in subjects with haplogroup T [HR 0.612 (95% CI 0.454, 0.824); P = 0.001] or in the superhaplogroup JT [HR 0.765 (95% CI 0.624, 0.938); P = 0.009]. Sensitivity analysis revealed that the results were robust. CONCLUSION: The mtDNA variants in the superhaplogroup JT associate with a reduced rate of radiographic OA progression. The mtDNA polymorphisms in the superhaplogroup JT emerge as potential complementary genetic biomarkers for disease progression.

Mitochondrial DNA haplogroups influence the risk of incident knee osteoarthritis in OAI and CHECK cohorts. A meta-analysis and functional study.

OBJECTIVE: To evaluate the influence of the mitochondrial DNA (mtDNA) haplogroups in the risk of incident knee osteoarthritis (OA) and to explain the functional consequences of this association to identify potential diagnostic biomarkers and therapeutic targets. METHODS: Two prospective cohorts contributed participants. The osteoarthritis initiative (OAI) included 2579 subjects of the incidence subcohort, and the cohort hip and cohort knee (CHECK) included 635, both with 8-year follow-up. The analysis included the association of mtDNA haplogroups with the rate of incident knee OA in subjects from both cohorts followed by a subsequent meta-analysis. Transmitochondrial cybrids harbouring haplogroup J or H were constructed to detect differences between them in relation to physiological features including specific mitochondrial metabolic parameters, reactive oxygen species production, oxidative stress and apoptosis. RESULTS: Compared with H, the haplogroup J associates with decreased risk of incident knee OA in subjects from OAI (HR=0.680; 95% CI 0.470 to 0.968; p<0.05) and CHECK (HR=0.728; 95% CI 0.469 to 0.998; p<0.05). The subsequent meta-analysis including 3214 cases showed that the haplogroup J associates with a lower risk of incident knee OA (HR=0.702; 95% CI 0.541 to 0.912; p=0.008). J cybrids show a lower free radical production, higher cell survival under oxidative stress conditions, lower grade of apoptosis as well as lower expression of the mitochondrially related pro-apoptotic gene BCL2 binding component 3 (BBC3). In addition, J cybrids also show a lower mitochondrial respiration and glycolysis leading to decreased ATP production. CONCLUSIONS: The physiological effects of the haplogroup J are beneficial to have a lower rate of incident knee OA over time. Potential drugs to treat OA could focus on emulating the mitochondrial behaviour of this haplogroup.

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.

iTRAQ-based analysis of progerin expression reveals mitochondrial dysfunction, reactive oxygen species accumulation and altered proteostasis.

INTRODUCTION: Nuclear accumulation of a mutant form of the nuclear protein Lamin-A, called Progerin (PG) or Lamin AΔ50, occurs in Hutchinson-Gilford Progeria Syndrome (HGPS) or Progeria, an accelerated aging disease. One of the main symptoms of this genetic disorder is a loss of sub-cutaneous fat due to a dramatic lipodystrophy. METHODS: We stably induced the expression of human PG and GFP -Green Fluorescent Protein- as control in 3T3L1 cells using a lentiviral system to study the effect of PG expression in the differentiation capacity of this cell line, one of the most used adipogenic models. Quantitative proteomics (iTRAQ) was done to study the effect of the PG accumulation. Several of the modulated proteins were validated by immunoblotting and real-time PCR. Mitochondrial function was analyzed by measurement of a) the mitochondrial basal activity, b) the superoxide anion production and c) the individual efficiency of the different complex of the respiratory chain. RESULTS: We found that over-expression PG by lentiviral gene delivery leads to a decrease in the proliferation rate and to defects in adipogenic capacity when compared to the control. Quantitative proteomics analysis showed 181 proteins significantly (p<0.05) modulated in PG-expressing preadipocytes. Mitochondrial function is impaired in PG-expressing cells. Specifically, we have detected an increase in the activity of the complex I and an overproduction of Superoxide anion. Incubation with Reactive Oxygen Species (ROS) scavenger agents drives to a decrease in autophagic proteolysis as revealed by LC3-II/LC3-I ratio. CONCLUSION: PG expression in 3T3L1 cells promotes changes in several Biological Processes, including structure of cytoskeleton, lipid metabolism, calcium regulation, translation, protein folding and energy generation by the mitochondria. Our data strengthen the contribution of ROS accumulation to the premature aging phenotype and establish a link between mitochondrial dysfunction and loss of proteostasis in HGPS.

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.