Estudio de mecanismos de comunicación celular en condrocitos articulares humanos: posible implicación en el desarrollo de la artrosis y búsqueda de una diana terapéutica

[Resumen] El cartílago articular es un tejido que recubre la superficie de los huesos en las articulaciones y permite el movimiento del esqueleto sin que se produzca dolor y fricción entre las superficies óseas. Los condrocitos articulares son las células responsables del mantenimiento del cartílago durante la vida adulta. La artrosis se caracteriza por la degeneración progresiva del cartílago articular. Los condrocitos se comunican entre sí a través de canales formados por conexina 43 (Cx43) y denominados Gap Junctions (GJs). La actividad y presencia de estos canales podrían ser esenciales para mantener la homeostasis y correcto funcionamiento del tejido modulando la comunicación célula-célula y célula-matriz. Además, la Cx43 posee múltiples funciones independientes de la actividad de los canales, y la sobreexpresión de Cx43 se correlacionó con la degeneración de la matriz extracelular del cartílago articular. En este trabajo de Máster hemos decidido investigar las funciones de la Cx43 en condrocitos de donantes sanos y artrósicos, con el objetivo de encontrar alguna alteración funcional que pueda explicar el cambio de fenotipo celular que ocurre en los condrocitos de los pacientes que sufren artrosis. Para ello, hemos diseñado un vector que contiene una secuencia de siRNA para disminuir la expresión de la Cx43 y un segundo vector para sobreexpresar este gen, pudiendo estudiar de esta manera los efectos de la Cx43 en expresión génica, proliferación y fenotipo celular. Por otro lado, también decidimos estudiar el efecto de la glicosilación de proteínas, incluida la Cx43, presentes en el cartílago y en la membrana de los condrocitos yrelacionadas con la adhesión y comunicación celular. Para ello utilizamosla lectina denominada MASL que se une específicamente a residuos terminales de ácido siálico α-2,3, modificación que ha sido relacionada con el desarrollo de artrosis y artritis reumatoide. Los resultados obtenidos demuestran que concentraciones nanomolaresde MASL protegen la estructura y composición del cartílago frente a un insulto o daño que desencadena la degradación de la matriz extracelularpor incremento de mediadores proinflamatorios o metaloproteasas.[Resumo] A cartilaxe articular é un tecido que recobre a superficie dos ósos nas articulacións e permite o movemento do esqueleto sen que se produza dor ou rozamento entre as superficies óseas. Os condrócitos articulares son as células responsables do mantemento da cartilaxe durante a vida adulta. A artrose caracterízase pola dexeneración progresiva da cartilaxe articular. Os condrócitos comunícanse entre sí a través dos canais formados por conexina 43 (Cx43) e denominados Gap Junctions (GJs). A actividade e presencia destos canais podrían ser esenciais para manter a homeostase e correcto funcionamento do tecido modulando a comunicación célula-célula e célula-matriz. Ademáis, a Cx43 posúe múltiples funcións independentes da actividade dos canais, e a sobreexpresión de Cx43 correlacionouse coa dexeneración da matriz extracelular da cartilaxe articular. Neste traballo de Mestrado decidimos investigar as funcións da Cx43 en condrócitos de donantes sáns e artrósicos, co obxectivo de encontrar algunha alteración funcional que poida explicar o cambio de fenotipo celular que ocorre nos condrócitos dos pacientes que sofren artrose. Para levalo a cabo, deseñamos un vector que contén a secuencia de siRNA para disminuir a expresión da Cx43 e un segundo vector para sobreexpresar este xene, poidendo estudar desta maneira os efectos da Cx43 na expresión xénica, proliferación e fenotipo celular. Por outro lado, tamén decidimos estudar o efecto da glicosilación de proteínas, incluida a Cx43 de proteínas, incluida a Cx43, presentes na cartilaxe e na membrana dos condrócitos e implicadas en adhesión e comunicación celular. Para facelo utilizamos a lectina chamada MASL que se une específicamente a residuos termináis de ácido siálico α-2,3, modificación que foi relacionada co desenvolvemento da artrose e artrite reumatoide. Os resultados obtidos demostran que concentracións nanomolares de MASL protexen a estructura e composición da cartilaxe fronte a un dano que da lugar a unha degradación da matriz extracelular por incremento de mediadores proinflamatorios ou metaloproteasas.[Abstract] Articular cartilage is a tissue that covers the surfaces of articular bones allowing the painless movement of the skeleton. Articular chondrocytes are the cells responsible for the synthesis and maintenance of cartilage during adult life. Osteoarthritis is characterized for the progressive degeneration of cartilage. Chondrocytes communicate each other through channels formed by connexin 43 (Cx43) called Gap Junctions (GJs). The presence of functional GJs channels might be essential to the maintenance of the homeostasis and proper functioning of the tissue by modulating the cell-cell and cell-matrix communication. Moreover, Cx43 has multiple functions independently of the activity of channels, and the overexpression of Cx43 was correlated with the degeneration of the extracellular matrix of articular cartilage. In this research work we have decided to investigate the functions of Cx43 in chondrocytes from healthy and osteoarthritic donors, with the goal of finding some functional impairment that might explain the change in the phenotype that it is observed in osteoarthritic chondrocytes. For this purpose, we have designed (i) a vector containing a siRNA sequence in order to reduce the Cx43 expression, and (ii) a vector to overexpress Cx43. We will investigate the role of Cx43 in gene expression, cell growth, proliferation and phenotype. Furthermore, we have also decided to study the effect of the glycosylation of glycoproteins, including Cx43, presented in the cartilage and in the membrane of chondrocytes, and related with cell adhesion and cell communication. We have used a lectin called MASL that specifically binds to terminal residues of α-2, 3 sialic acid, modification that has been previously associated with the development of osteoarthritis and rheumatoid arthritis. The results presented here show that nanomolar concentrations of MASL can preserve cartilage structure when cartilage or primary chondrocytes are treated with oligomycin, which triggers the extracellular matrix degradation through the increase of inflammatory cytokines or metalloproteinase.Traballo fin de mestrado (UDC.FCS). Asistencia e investigación sanitaria. Especialidade en fundamentos de investigación biomédica. Curso 2013/201

Senolytic Activity of Small Molecular Polyphenols from Olive Restores Chondrocyte Redifferentiation and Promotes a Pro-Regenerative Environment in Osteoarthritis

[Abstract] Articular cartilage and synovial tissue from patients with osteoarthritis (OA) show an overactivity of connexin43 (Cx43) and accumulation of senescent cells associated with disrupted tissue regeneration and disease progression. The aim of this study was to determine the effect of oleuropein on Cx43 and cellular senescence for tissue engineering and regenerative medicine strategies for OA treatment. Oleuropein regulates Cx43 promoter activity and enhances the propensity of hMSCs to differentiate into chondrocytes and bone cells, reducing adipogenesis. This small molecule reduce Cx43 levels and decrease Twist-1 activity in osteoarthritic chondrocytes (OACs), leading to redifferentiation, restoring the synthesis of cartilage ECM components (Col2A1 and proteoglycans), and reducing the inflammatory and catabolic factors mediated by NF-kB (IL-1ß, IL-6, COX-2 and MMP-3), in addition to lowering cellular senescence in OACs, synovial and bone cells. Our in vitro results demonstrate the use of olive-derived polyphenols, such as oleuropein, as potentially effective therapeutic agents to improve chondrogenesis of hMSCs, to induce chondrocyte re-differentiation in OACs and clearing out senescent cells in joint tissues in order to prevent or stop the progression of the disease.Xunta de Galicia; IN607B 2017/21Xunta de Galicia; ED481A-2015/188Xunta de Galicia; IN606B-2019/004Xunta de Galicia; IN606B-2017/014This work was supported in part through funding from the Spanish Foundation for Research on Bone and Mineral Metabolism (FEIOMM), grant PRECIPITA-2015-000139 from the FECYT-Ministry of Economy and Competitiveness (to M.D.M.), grant PI16/00035 and PI19/00145 from the Health Institute ‘Carlos III’ (ISCIII, Spain), the European Regional Development Fund, ‘A way of making Europe’ from the European Union (to M.D.M.) and a grant from Xunta de Galicia IN607B 2017/21 (to M.D.M.). M.V.-E. was funded with a predoctoral (ED481A-2015/188) and a postdoctoral fellowship (IN606B-2019/004) from Xunta de Galicia. P.C.-F. was funded with a postdoctoral fellowship (IN606B-2017/014) from Xunta de Galicia

Targeting of chondrocyte plasticity via connexin43 modulation attenuates cellular senescence and fosters a pro-regenerative environment in osteoarthritis

Osteoarthritis (OA), a chronic disease characterized by articular cartilage degeneration, is a leading cause of disability and pain worldwide. In OA, chondrocytes in cartilage undergo phenotypic changes and senescence, restricting cartilage regeneration and favouring disease progression. Similar to other wound-healing disorders, chondrocytes from OA patients show a chronic increase in the gap junction channel protein connexin43 (Cx43), which regulates signal transduction through the exchange of elements or recruitment/release of signalling factors. Although immature or stem-like cells are present in cartilage from OA patients, their origin and role in disease progression are unknown. In this study, we found that Cx43 acts as a positive regulator of chondrocyte-mesenchymal transition. Overactive Cx43 largely maintains the immature phenotype by increasing nuclear translocation of Twist-1 and tissue remodelling and proinflammatory agents, such as MMPs and IL-1β, which in turn cause cellular senescence through upregulation of p53, p16INK4a and NF-κB, contributing to the senescence-associated secretory phenotype (SASP). Downregulation of either Cx43 by CRISPR/Cas9 or Cx43-mediated gap junctional intercellular communication (GJIC) by carbenoxolone treatment triggered rediferentiation of osteoarthritic chondrocytes into a more differentiated state, associated with decreased synthesis of MMPs and proinflammatory factors, and reduced senescence. We have identified causal Cx43- sensitive circuit in chondrocytes that regulates dedifferentiation, redifferentiation and senescence. We propose that chondrocytes undergo chondrocyte-mesenchymal transition where increased Cx43-mediated GJIC during OA facilitates Twist-1 nuclear translocation as a novel mechanism involved in OA progression. These findings support the use of Cx43 as an appropriate therapeutic target to halt OA progression and to promote cartilage regeneration.This work was supported in part through funding from the Spanish Society for Rheumatology (SER; FER 2013) and Spanish Foundation for Research on Bone and Mineral Metabolism (FEIOMM), grant PRECIPITA-2015-000139 from the FECYT-Ministry of Economy and Competitiveness (to M.D.M.), grant PI16/00035 from the Health Institute ‘Carlos III’ (ISCIII, Spain), the European Regional Development Fund, ‘A way of making Europe’ from the European Union (to M.D.M.) and a grant from Xunta de Galicia IN607B 2017/21 (to M.D.M.) and pre-doctoral fellowship to M.V.-E. T.A. acknowledges support from Instituto de Salud Carlos III grants PI16/00772 and CPII16/00042, co-financed by the European Regional Development Fund (ERDF)S

Extracellular Vesicles Enriched in Connexin 43 Promote a Senescent Phenotype in Bone and Synovial Cells Contributing to Osteoarthritis Progression

[Abstract] The accumulation of senescent cells is a key characteristic of aging, leading to the progression of age-related diseases such as osteoarthritis (OA). Previous data from our laboratory has demonstrated that high levels of the transmembrane protein connexin 43 (Cx43) are associated with a senescent phenotype in chondrocytes from osteoarthritic cartilage. OA has been reclassified as a musculoskeletal disease characterized by the breakdown of the articular cartilage affecting the whole joint, subchondral bone, synovium, ligaments, tendons and muscles. However, the mechanisms that contribute to the spread of pathogenic factors throughout the joint tissues are still unknown. Here, we show for the first time that small extracellular vesicles (sEVs) released by human OA-derived chondrocytes contain high levels of Cx43 and induce a senescent phenotype in targeted chondrocytes, synovial and bone cells contributing to the formation of an inflammatory and degenerative joint environment by the secretion of senescence-associated secretory associated phenotype (SASP) molecules, including IL-1ß and IL-6 and MMPs. The enrichment of Cx43 changes the protein profile and activity of the secreted sEVs. Our results indicate a dual role for sEVs containing Cx43 inducing senescence and activating cellular plasticity in target cells mediated by NF-kß and the extracellular signal-regulated kinase 1/2 (ERK1/2), inducing epithelial-to-mesenchymal transition (EMT) signalling programme and contributing to the loss of the fully differentiated phenotype. Our results demonstrated that Cx43-sEVs released by OA-derived chondrocytes spread senescence, inflammation and reprogramming factors involved in wound healing failure to neighbouring tissues, contributing to the progression of the disease among cartilage, synovium, and bone and probably from one joint to another. These results highlight the importance for future studies to consider sEVs positive for Cx43 as a new biomarker of disease progression and new target to treat OA.This work was supported in part through funding from Health Institute ‘Carlos III’ (ISCIII, Spain), the European Regional Development Fund, ‘A way of making Europe’ from the European Union (to MDM): grant PI19/00145; a grant from the Joint Transnational Call for Proposals for “European Innovative Research & Technological Development Projects in Nanomedicine” EURONANOMED III (AC21_2/00026) (to MDM); a grant from Xunta de Galicia (IN607B2020/12) (to MDM) and from H2020, Future and Emerging Technologies (grant 858014 “PANACHE”) to MDM. MV-E was funded with a predoctoral (ED481A-2015/188) and post-doctoral fellowship (IN606B-2019/004) from Xunta de Galicia. AG-C was funded with a predoctoral fellowship (FIS20/00310) from ISCIII. PC-F was funded with a post-doctoral fellowship and a grant from Xunta de Galicia (INB606B 2017/014 and IN606C 2021/006). We thank members of the CellCOM group for helpful technical suggestion, María Dolores Álvarez Alvariño (CHUS) for generously collecting tissue samples in the operating room after surgery and Arantxa Tabernero (INCYL, University of Salamanca) for kindly providing the human Cx43 plasmid used in this studyXunta de Galicia; IN607B2020/12Xunta de Galicia; ED481A-2015/188Xunta de Galicia; IN606B-2019/004Xunta de Galicia; INB606B 2017/014Xunta de Galicia; IN606C 2021/00

Role of Connexin43 in Cell Plasticity and Tissue Degeneration in Osteoarthritis

Programa Oficial de Doutoramento en Bioloxía Celular e Molecular . 5004V01[Resumen] La artrosis (OA) es una enfermedad crónica caracterizada por la degradación progresiva del cartílago articular que supone una de las mayores causa de discapacidad y dolor en el mundo. Los condrocitos en el cartílago de pacientes con OA (OAc) sufren cambios en el fenotipo celular que interfieren con la regeneración del cartílago y favorecen el progreso de la enfermedad. De manera similar a lo que se ha descrito en alteraciones en el proceso reparación en otros tejidos, los OAc presentan niveles elevados de una proteína transmembrana denominada conexina43 (Cx43), que participa en la comunicación intercelular y en la regulación de diferentes vías de señalización a través del reclutamiento/liberación de factores de señalización. Los resultados de este trabajo demuestran que la Cx43 activa el proceso de desdiferenciación de los condrocitos adultos a un estado más inmaduro. La sobreactividad de la Cx43 mantiene este fenotipo inmaduro mediante el incremento de la translocación nuclear de Twist-1. La activación de Twist-1 activa rutas implicadas en desdiferenciación celular lo que aumenta los índices de proliferación de las células inmaduras, síntesis de factores de remodelado de la matriz extracelular y agentes proinflamatorios como MMPs e IL-1ß, respectivamente. Esta activación desencadena en un aumento en los niveles de senescencia celular mediada por p53/p16INK4a y activación de NF-kß, lo que contribuye al fenotipo secretor asociado a senescencia (SASP). La Cx43 modifica el contenido y función de las vesículas extracelulares (VEs), vía anclaje de componentes como proteínas y moléculas de ARN o ADN a sus dominios citoplasmáticos y extracelulares. Las VEs positivas para Cx43 activan la desdiferenciación celular en las células vecinas, amplificando la función de la Cx43 en plasticidad celular a células cercanas y distantes. La disminución en los niveles de Cx43 y de sus canales mediante CRISPR-Cas9, así como el uso de pequeñas moléculas como la oleuropeína o péptidos miméticos, desencadenaron en la rediferenciación de los OAc a un estado adulto diferenciado asociado con una menor producción de MMPs y expresión de factores proinflamatorios como IL-1ß, disminuyendo senescencia celular. Los resultados demuestran que la Cx43 controla la plasticidad celular de condrocitos articulares identificando una vía de control de desdiferenciación/rediferenciación y senescencia, eventos críticos durante el proceso de reparación o regeneración tisular. Estos hallazgos indican que la Cx43 sería una diana terapéutica apropiada para evitar el desarrollo o progresión de la artrosis. La modulación de la actividad de esta proteína podría ayudar a diseñar terapias celulares más eficientes con el objetivo de promover la regeneración del cartílago articular.[Resumo] A artrose é unha enfermidade caracterizada pola dexeneración da cartilaxe articular que supón unha das maiores causas de discapacidade e dor no mundo. Os condrócitos artrósicos (OAc) sofren cambios de fenotipo celular que impiden a rexeneración da cartilaxe e favorecen a progresión da enfermidade. De maneira similar ao descrito en alteracións no proceso de reparación noutros tecidos, os OAc presentan niveles elevados dunha proteína transmembrana denominada conexina43 (Cx43), que participa na comunicación intercelular e na regulación de diferentes vías de sinalización a través do recrutramento/liberación de factores de sinalización. Os resultados deste estudo demostraron que a Cx43 activa o proceso de desdiferenciación dos condrócitos adultos a un estado máis inmaduro. A sobreactivación da Cx43 mantén este fenotipo inmaduro mediante o incremento da tranlocación nuclear de Twist-1. A activación de Twist-1 activa rutas implicadas en desdiferenciación celular, o que aumenta os índices de proliferación das células inmaduras, síntese de factores remodeladores da matriz extracelular e axentes proinflamatorios, como MMPs e IL-1ß, respectivamente. Esta activación desencadena nun aumento nos niveles de senescencia celular mediada por p53/p16INK4a e activación de NF-kß, o que contribúe ao fenotipo secretor asociado a senescencia (SASP). A Cx43 pode modificar o contido e función das vesículas extracelulares (VEs), a través da ancoraxe de componentes coma proteínas e moléculas de ARN ou ADN aos seus dominios citoplasmáticos e extracelulares. As VEs, positivas para Cx43, activan a desdiferenciación celular en células veciñas, amplificando a función da Cx43 en plasticidade celular a células cercanas e distantes. A disminución nos niveis de Cx43 e das súas canles mediante CRISPR-Cas9 ou o uso de pequenas moléculas como a oleuropeína ou péptidos miméticos, desencadenaron na rediferenciación dos OAc a un estado adulto diferenciado asociado cunha menor producción de MMPs e expresión de factores proinflamatorios, como IL-1ß, disminuindo a senescencia celular. Os resultados demostran que a Cx43 controla a plasticidade celular en condrócitos articulares, identificando unha vía de control de desdiferenciación/rediferenciación e senescencia, eventos críticos durante o proceso de reparación ou rexeneración tisular. Estos achados indican que a Cx43 sería una diana terapéutica apropiada para evitar o desenvolvemento ou progresión da artrose. A modulación da actividade desta proteína podería axudar a deseñar terapias celulares máis eficientes co obxectivo de promover a rexeneración da cartilaxe articular.[Abstract] Osteoarthritis (OA) is a chronic disease and a leading cause of disability and pain worldwide, characterized by articular cartilage degradation and joint degeneration. OA chondrocytes (OAc) undergo phenotypic changes and senescence, restricting cartilage regeneration and favouring disease progression. Similar to other wound-healing disorders, OAc show a chronic increase in the transmembrane channel protein connexin43 (Cx43), which regulates signal transduction through the exchange of elements or recruitment/release of signalling factors. The results of this study demonstrated that Cx43 acts as a positive regulator of human chondrocyte reversion to a less differentiated state. Overactive Cx43 maintains this immature phenotype by increasing the nuclear translocation of Twist-1 and increasing the expression of tissue remodelling and proinflammatory agents, such as MMPs and IL-1β, which finally results in cellular senescence through upregulation of p53/p16INK4a and NF-κB, contributing to the senescence-associated secretory phenotype (SASP). In addition, Cx43 can modify the content of the extracellular vesicles probably by facilitating the binding of proteins and molecules of RNA and DNA involved in different signalling routes. The results indicate that the deliver of this content seems to control cellular plasticity in nearby and distant cells. However, downregulation of Cx43 (in chondrocytes and EVs), using the small molecule oleuropein or a mimetic peptide, led to rediferentiation of OAc into a more differentiated state, associated with decreased production of MMPs and expression of proinflammatory factors, such as IL-1ß. This effect results in a reduction in cellular senescence. Collectively, these results identify a causal Cx43-sensitive circuit in chondrocytes that regulates dedifferentiation/redifferentiation and senescence, events critical to wound healing and tissue regeneration. Targeting Cx43 allows immature chondrocytes to revert to a chondrocyte-specific phenotype and its subsequent recovery in a predictable manner. These findings support the use of Cx43 as an appropriate therapeutic target to halt OA progression and to promote cartilage regeneration

Cellular senescence

Cellular senescence defines a state of stable and generally irreversible proliferative arrest associated with various morphological, structural and functional changes (Figure 1), including enhanced expression and secretion of pro-inflammatory and tissue-remodelling mediators. This state is crucial in tissue physiology and pathology and arises as a response to potentially damaging stress signals. Whether the activation of a senescence state provides benefits or detriments for tissue function and homeostasis is strictly dependent on the context. Cell senescence acts as a potent tumour-suppressive mechanism limiting the proliferation of cells at risk of malignant transformation and supports the repair of acute tissue damage, but also represents a key driver of ageing and age-related diseases

Cellular senescence

Cellular senescence defines a state of stable and generally irreversible proliferative arrest associated with various morphological, structural and functional changes (Figure 1), including enhanced expression and secretion of pro-inflammatory and tissue-remodelling mediators. This state is crucial in tissue physiology and pathology and arises as a response to potentially damaging stress signals. Whether the activation of a senescence state provides benefits or detriments for tissue function and homeostasis is strictly dependent on the context. Cell senescence acts as a potent tumour-suppressive mechanism limiting the proliferation of cells at risk of malignant transformation and supports the repair of acute tissue damage, but also represents a key driver of ageing and age-related diseases