Identificación de moléculas reguladoras del envejecimiento articular para el tratamiento de la artrosis

Programa Oficial de Doctorado en Ciencias de la Salud. 5007V01[Resumo] Os fallos nos mecanismos de homeostase relacionados coa idade, como o defecto de autofaxia e a acumulación de células senescentes, contribúen á dexeneración da cartilaxe e á artrose (OA), para a cal no existen fármacos modificadores da enfermidade. O noso obxectivo desta Tesis Doutoral foi identificar compostos reguladores de ambos procesos para o tratamento da OA. Para iso, os condrócitos humanos foron tratados con IL-6, unha citoquina pro-inflamatoria presente no secretoma das células en estado de senescencia, para inducir senescencia e inhibir o fluxo de autofaxia. Para a realización dun cribado de alto rendemento con compostos da quimioteca de reposicionamento Prestwick de medicamentos aprobados, a actividade asociada á senescencia β-Galactosisada (SA-β-Gal) e o marcador LC3 foron empregados como marcadores de senescencia e de fluxo de autofaxia, respectivamente. Como resultado identificáronse 14 compostos con actividade senoterapéutica e pro-autofáxica, dos cales escolleuse para a súa confirmación o Fenofibrato (FN), un fibrato e agonista PPARα empleado no tratamento das dislipemias en humanos, para a súa confirmación dada a relevancia do seu mecanismo de acción metabólico. Modelos preclínicos celulares, tisulares e de sangue de pacientes artrósicos e envellecidos foron empregados para testar a eficacia e a relevancia de activar PPARα para o tratamento da OA. Os resultados mostraron que o FN eliminou selectivamente as células senescentes a través da vía de apoptose, incrementou o fluxo de autofaxia e protexeu fronte a degradación da cartilaxe. Ademais, a expresión de PPARα redúxse en ratóns envellecidos e artrósicos e en sangue e na cartilaxe procedente de pacientes con OA de rodilla. É importante destacar que nun estudio retrospectivo en pacientes con OA de rodilla procedentes da cohorte americana Osteoarthritis Initiative (OAI), o tratamento con fibratos mellorou significativamente as condicións clínicas. Estes resultados demostran que os fibratos, fármacos aprobados para o tratamento de alteracións no metabolismo lipídico, poderían ter unha inmediata utilidade clínica para o tratamento do envellecemento articular e da OA.[Resumen] Los fallos en los mecanismos de homeostasis relacionados con la edad, como el defecto de autofagia y la acumulación de células senescentes, contribuyen a la degeneración del cartílago y a la artrosis (OA), para la cuál no existen fármacos modificadores de la enfermedad. Nuestro objetivo es esta Tesis Doctoral fue identificar compuestos reguladores de ambos procesos para el tratamiento de la OA. Para ello, los condrocitos humanos fueron tratados con IL-6, una citoquina proinflamatoria presente en el secretoma de las células en estado de senescencia, para inducir senescencia e inhibir el flujo de autofagia. Para la realización de un cribado de alto rendimiento con compuestos de la quimioteca de reposicionamiento Prestwick de medicamentos aprobados, la actividad asociada a senescencia β-Galactosisada (SA- β-Gal) y el marcador LC3 fueron empleados como marcadores de senescencia y de flujo de autofagia, respectivamente. Como resultado se identificaron 14 compuestos con actividad senoterapéutica y pro-autofágica, de los cuales se escogió el Fenofibrato (FN), un fibrato y agonista PPARα empleado en el tratamiento de las dislipemias en humanos, para su confirmación dada la relavancia de su mecanismo de acción metabólico. Modelos preclínicos celulares, tisulares y de sangre de pacientes artrósicos y envejecidos fueron empleados para testar la eficacia y la relevancia de activar PPARα para el tratamiento de la OA. Los resultados mostraron que el FN eliminó selectivamente las células senescentes a través de la vía de apoptosis, incrementó el flujo de autofagia y protegió frente a la degradación del cartílago. Además, la expresión de PPARα se redujo en ratones envejecidos y artrósicos y en sangre y en cartílago procedente de pacientes con OA de rodilla. Es importante destacar que en un estudio retrospectivo en pacientes con OA de rodilla procedentes de la cohorte americana Osteoarthritis Initiative (OAI), el tratamiento con fibratos mejoró significativamente las condiciones clínicas. Estos resultados demuestran que los fibratos, fármacos aprobados para el tratamiento de alteraciones en el metabolismo lipídico, podrían tener una inmediata utilidad clínica para el tratamiento del envejecimiento articular y la OA.[Abstract] Ageing-related failure of homeostasis mechanisms, such as defects of autophagy and accumulation of senescent cells, contributes to articular cartilage degeneration and osteoarthritis (OA), for which disease-modifying treatments are not available. Our objective in this Thesis Doctoral was to identify compounds to prevent OA by regulating chondrocyte senescence and autophagy. For that, human chondrocytes were treated with IL-6, a proinflammatory cytokine present in the secretome of senescent cells, to induce senescence and inhibit autophagy flux. For high-throughtput screening with compounds from the Prestwick Chemical Library of approved drugs, the activity of senescence associated β-Galactosidase (SA-β-gal) and LC3 marker were used as marker of senescence and autophagic flux, respectively. As a result, 14 compounds were identified with senotherapeutic and pro-autophagic activity, of which Fenofibrate (FN), a fibrate and PPARα agonist used in the treatment of dyslipidemia in humans, was chosen for confirmation given the relavance of its mechanism of metabolic action. Preclinical cellular, tissue and blood from OA and blood from OA and ageing models were used to test the efficacy and relevance of activating PPARα for the treatment of OA. The results showed that FN selectively eliminated senescent cells via apoptosis, increased autophagic flux and protected against cartilage degradation. Moreover, PPARα expression was reduced through both ageing and OA in mice and also in blood and cartilage from knees of OA patients. Remarkably, in a retrospective study, in human patients with knee OA from the Osteoarthritis Initiative (OAI) Cohort, fibrate treatment improved OA clinical conditions. These results demonstrate that fibrates, approved drugs targeting lipid metabolism, could have immediate clinically utility for age-related cartilage degeneration and OA treatment

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

Fibrates as drugs with senolytic and autophagic activity for osteoarthritis therapy

Research paper[Abstract] Background. Ageing-related failure of homeostasis mechanisms contributes to articular cartilage degeneration and osteoarthritis (OA), for which disease-modifying treatments are not available. Our objective was to identify molecules to prevent OA by regulating chondrocyte senescence and autophagy. Methods. Human chondrocytes with IL-6 induced senescence and autophagy suppression and SA-β-gal as a reporter of senescence and LC3 as reporter of autophagic flux were used to screen the Prestwick Chemical Library of approved drugs. Preclinical cellular, tissue and blood from OA and blood from OA and ageing models were used to test the efficacy and relevance of activating PPARα related to cartilage degeneration. Findings. Senotherapeutic molecules with pro-autophagic activity were identified. Fenofibrate (FN), a PPARα agonist used for dyslipidaemias in humans, reduced the number of senescent cells via apoptosis, increased autophagic flux, and protected against cartilage degradation. FN reduced both senescence and inflammation and increased autophagy in both ageing human and OA chondrocytes whereas PPARα knockdown conferred the opposite effect. Moreover, PPARα expression was reduced through both ageing and OA in mice and also in blood and cartilage from knees of OA patients. Remarkably, in a retrospective study, fibrate treatment improved OA clinical conditions in human patients from the Osteoarthritis Initiative (OAI) Cohort. Interpretation. These results demonstrate that FDA-approved fibrate drugs targeting lipid metabolism protect against cartilage degeneration seen with ageing and OA. Thus, these drugs could have immediate clinically utility for age-related cartilage degeneration and OA treatment.Instituto de Salud Carlos III; PI14/01324Instituto de Salud Carlos III; PI17/02059Ministerio de Economía y Competitividad; P01 AG043376Ministerio de Economía y Competitividad; U19 AG05627