Cíbridos transmitocondriales como modelo in vitro para estudiar la asociación existente entre el ADN mitocondrial y la patología artrósica

Programa Oficial de Doutoramento en Ciencias da Saúde. 5007V01[Resumen] La artrosis es una patología compleja que se origina debido a la pérdida del equilibrio entre los procesos anabólicos y catabólicos, desencadenando la modificación anatómica y la alteración fisiológica de la función articular. En los últimos años se ha establecido una relación entre la función mitocondrial, su genoma y la incidencia y/o progresión de esta patología. Hoy en día el uso de modelos celulares como los cíbridos transmitocondriales que se caracterizan por presentar un mismo fondo nuclear pero diferente genoma mitocondrial, resultan esenciales para estudiar el papel de la mitocondria en patologías complejas como la artrosis. El objetivo de esta tesis fue la generación de células carentes de genoma mitocondrial, así como la generación de cíbridos portadores de los haplogrupos de ADNmt H y J de individuos sanos y artrósicos para analizar de qué manera la función mitocondrial está relacionada con mecanismos que conllevan a la alteración del cartílago articular. El análisis de parámetros relacionados con el desarrollo de esta patología como el estrés oxidativo, apoptosis, autofagia o el metabolismo glucolítico y lipídico, nos permitió demostrar la relevancia de las mitocondrias funcionales en la fisiopatología de la artrosis. Los resultados obtenidos pusieron de manifiesto la existencia de diferencias entre los cíbridos portadores de diferentes ADN mitocondriales tanto a nivel funcional como molecular.[Resumo] A artrose é unha patoloxía complexa que se orixina debido á perda do equilibrio entre os procesos anabólicos e catabólicos, desencadeando a modificación anatómica e a alteración fisiolóxica da función articular. Nos últimos anos, estableceuse unha relación entre a función da mitocondria, o seu xenoma e a incidencia e/o progresión de esta patoloxía. Hoxe en día, o uso de modelos celulares como os cíbridos transmitocondriais que caracterízanse por ter o mesmo fondo nuclear pero diferente xenoma mitocondriai, permiten estudar o papel da mitocondria en patoloxías complexas coma a artrose. O obxectivo desta tese foi a xeración de células carentes de xenoma mitocondriai e a xeración de cíbridos portadores dos haplogrupos H e J de individuos sans e artríticos para analizar de que maneira a función mitocondriai pódese relacionar cos mecanismos que levan á alteración da cartilaxe articular. A análise de parámetros que relacionáronse có desenvolvemento desta patoloxía coma o estrés oxidábel, apoptose, autofaxia ou o metabolismo da glucosa e dos lípidos, demostrou a relevancia das mitocondrias funcionais na fisiopatoloxía da artrose. Os resultados presentados nesta tese puxeron de manifesto as diferenzas entre os cíbridos orixinados a partir de diferentes xenomas mitocondriais tanto ó nivel funcional coma molecular.[Abstract] Osteoarthritis is a complex disease in which an imbalance between anabolic and catabolic processes leads to the anatomical and physiological alteration of the joint function. During the last years, mitochondrial function and genome have been associated with the incidence and/or progression of osteoarthritis. Nowadays, cell models such as transmitochondrial cybrids, which have the same nuclear background but different mitochondrial genome, are a useful tool to study the role of mitochondria in complex diseases like osteoarthritis. The aim of this thesis was to generate cells without mitochondrial genome and to establish transmitochondrial cybrids carrying the H and J haplogroups from healthy and osteoarthritic donors. This allowed us to study how the mitochondrial function may be related to mechanisms that initiate molecular alterations at the joint. Analyzing the parameters that have been related to osteoarthritis development, like oxidative stress, apoptosis, autophagy, glucose and fatty acid metabolism, we could demonstrate the relevance of functional mitochondria in the pathophysiology of osteoarthritis. To conclude, the results presented in this thesis showed that cybrids carrying different mitochondria genomes responded differently at functional and molecular levels

Impaired metabolic flexibility in the osteoarthritis process: a study on transmitochondrial cybrids

[Abstract] 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 β-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.Instituto de Salud Carlos III; CB06/01/0040Instituto de Salud Carlos III; RETIC-RIER-RD16/0012/0002Instituto de Salud Carlos III; PI12/00329Instituto de Salud Carlos III; PI14/01254Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; PRB3-ISCIII-PT17/0019Instituto de Salud Carlos III; PI17/0021

Oleate prevents palmitate-induced mitochondrial dysfunction in chondrocytes

[Abstract] 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.Instituto de Salud Carlos III; PI14/01254Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; RETIC-RIER-RD16/0012/0002Instituto de Salud Carlos III; PRB3-ISCIII-PT17/0019/0014Xunta de Galicia; IN607A2017/1

Mitochondrial DNA (mtDNA) haplogroups J and H are differentially associated with the methylation status of articular cartilage: potential role in apoptosis and metabolic and developmental processes

[Abstract] 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. GSE 43269). 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 qB asePlus 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.Instituto de Salud Carlos III; CIBERCB06/01/0040‐SpainInstituto de Salud Carlos III; CPII17/00026Instituto de Salud Carlos III; PI14/01254Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; PI17/0021

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-1β 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

Generating Rho-0 Cells Using Mesenchymal Stem Cell Lines.

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

[Abstract] 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.Instituto de Salud Carlos III; CB06/01/0040Instituto de Salud Carlos III; RETIC-RIER-RD16/0012/0002Instituto de Salud Carlos III; PI12/00329Instituto de Salud Carlos III; PI14/01254Instituto de Salud Carlos III; PI16/02124Instituto de Salud Carlos III; PIE16/00054Instituto de Salud Carlos III; PRB3-ISCIII-PT17/0019Instituto de Salud Carlos III; PI17/00210Instituto de Salud Carlos III; CPII17/0002

Characterization of 3a6 cells without treatment (3a6-wt) and 3a6 Rho-0 like cells.

<p>(<b>a</b>). Phenotypic characterization is represented as the percentage of positivity for CD29, CD73, CD90, CD105, CD166 and SSEA4 for both cell types. (<b>b</b>). Cellular levels of reactive oxygen species (ROS) in both cell types and in 143B.TK-Rho-0 cells (used as a reference for a typical Rho-0 line). Total ROS production was measured with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA); data are expressed as mean fluorescence intensity. (<b>c</b>). Apoptosis measured with Annexin-V-FIT: data are expressed as percentage of positive cells for Annexin-V and propidium iodide (PI) in basal conditions and culture in presence of Staurosporine (Stau) at 2 μM for 2 hours. (<b>d</b>). Mitochondrial membrane potential (Δψm) measured with DilC1(5) [1,1´,3,3,3´-hexamethylindodicarbo-cyanine iodide], data are expressed as percentages of cells that were positive for DilC1(5) fluorescence. (<b>e</b>). Mitochondrial network in 3a6-wt and 3a6-Rho 0 cells incubated with 250 nM MitoTraker Red solution for 30 min in a 37°C incubator. The cells were fixed with 4% paraformaldehyde and counterstained with Hoechst-33258 nuclear dye. The cells were photographed with a confocal microscope Nikon AR-1. (<b>f-g</b>) The mitochondrial respiration [oxygen consumption (OCR)] pattern was obtained using a SeaHorse XFp for 3a6-wt and 3a6 Rho-0 cells (<b>f</b>) and for 143B.TK- and 143B.TK-Rho-0 cells (<b>g</b>). All data were obtained from three independent experiments, expressed as mean ±SD and analyzed by the unpaired t-test (*, p≤0.05; *** p<0.001).</p

Summary of the most effective reagent reducing mitochondrial DNA (mtDNA) copy number in 3a6 human mesenchymal stem cells (hMSCs).

<p>Summary of the most effective reagent reducing mitochondrial DNA (mtDNA) copy number in 3a6 human mesenchymal stem cells (hMSCs).</p

3a6 cell line response to different substances capable of depleting the levels of mitochondrial DNA.

<p>All mtDNA copy numbers are expressed as percentages comparing each value with untreated cells valued at 100% (<b>a</b>). Treatment with ethidium bromide (EtBr) at three different concentrations (1 mM, 100 nM and 500 nM) for 240 hours. The maximum effectiveness of this treatment was at 100 nM. (<b>b</b>). Treatment with Rhodamine 6g at 1, 3 and 5 μg/ml for 24, 72, 120 and 168 hours. (<b>c</b>). Treatment with 1-methyl-4-phenylpyridinium (MPP⁺) at 25 μM, 0.5 mM and 1 mM for 72 hours. (<b>d</b>). Treatment with Zidovudine (AZT) at 20 μM and 0.5 mM, for 96, 144 and 216 hours. AZT increased mtDNA content. (<b>e</b>). Treatment with Stavudine (d4t) at 5, 10 and 100 μM and 0.5 mM for 96 and 144 hours. The analysis of mtDNA copy numbers reflect that treatment with the highest concentration (0.5 mM) for 144 hours decreased the levels of mtDNA in treated cells nearly 98% compared to those of untreated cells. 8x10⁴ cells were plated in each experiment for treatment with a reagent. The figures represent at least three independent experiments.</p