Role of p65/RelA in cellular senescence and inflamation

[Resumen]: El factor de transcripción NF-kβ es una familia de proteínas relacionada con multitud de eventos celulares, entre los que destacan la inflamación, el cáncer y la senescencia celular, por lo que la descripción de los mecanismos de activación y silenciamiento de esta ruta es esencial para el conocimiento acerca de estos procesos. El principal componente de NF-kβ es p65, el cual, es un activador de SASP. Este SASP está estrechamente relacionado con los procesos de la senescencia celular e inflamación, por lo que en este trabajo nos planteamos, mediante un desarrollo experimental basado en estudios moleculares, entender el papel de p65 en dichos procesos mediante su inducción en MSCs y la inhibición del la misma. Además, mediante la inhibición de p65 (usando MG-132, curcumina y JSH-23), no solo hemos podido validar su relación con la senescencia y la inflamación, si no, que la acción de estos inhibidores pone de relevancia su uso como senomórficos, con el fin de influir en futuras terapias celulares asociadas a paliar los efectos de la senescencia celular e inflamación.[Resumo]: O factor de transcripción NF-kβ é unha familia de proteínas relacionadas cunha multitude de eventos celulares, entre os que destaca a inflamación, o cancro e a senescencia celular, polo que a descripción dos mecanismos de activación e silenciamento desta ruta é esencial para o coñecemento de ditos procesos. O compoñent principal de NF-kβ é p65, o cal, é un activador de SASP. Iste SASP está estreitamente relacionado cos procesos de senescencia celular e inflamacuón, polo qué niste traballo planteamonos, mediante un desenvolvemento experimental basado en estudos moleculares, entender o papel de p65 en ditos procesos mediante a inhibición de p65 (MG-132, curcumina e JSH-23), non só puidemos validar a súa releción coa senescencia e a inflamación, se non, que a acción destes inhibidores pon de relieve o seu uso como senomórficos, có fin de influir en furturas terapias celulares asociadas a paliar os efectos da senescencia celular e a inflamación.[Abstract]: The transcription factor NF-β is a family of proteins related to a multitude of cellular events, including inflammation, cancer and cellular senescence, so the description of the mechanisms of activation and silencing of this pathway is essential for the understanding of these processes. The main component of NF-β is p65, which is an activator of SASP. This SASP is closely related to the processes of cellular senescence and inflammation, so in this work we aimed to know the role of p65 in these processes by means of an experimental development based on molecular studies and its induction in MSCs and its inhibition. Furthermore, by inhibiting p65 (MG-132, curcumin and JSH-23), we have not only been able to validate its relationship with senescence and inflammation, but the action of these inhibitors highlights its use as a senomorphic, in order to influence future cell therapies associated with alleviating the effects of cellular senescence and inflammation.Traballo fin de mestrado (UDC.CIE). Bioloxía molecular, celular e xenética. Curso 2020/202

Therapeutic Potential for Regulation of the Nuclear Factor Kappa-B Transcription Factor p65 to Prevent Cellular Senescence and Activation of Pro-Inflammatory in Mesenchymal Stem Cells

[Abstract] Mesenchymal stem cells have an important potential in the treatment of age-related diseases. In the last years, small extracellular vesicles derived from these stem cells have been proposed as cell-free therapies. Cellular senescence and proinflammatory activation are involved in the loss of therapeutic capacity and in the phenomenon called inflamm-aging. The regulators of these two biological processes in mesenchymal stem cells are not well-known. In this study, we found that p65 is activated during cellular senescence and inflammatory activation in human umbilical cord-derived mesenchymal stem cell. To demonstrate the central role of p65 in these two processes, we used smallmolecular inhibitors of p65, such as JSH-23, MG-132 and curcumin. We found that the inhibition of p65 prevents the cellular senescence phenotype in human umbilical cord-derived mesenchymal stem cells. Besides, p65 inhibition produced the inactivation of proinflammatory molecules as components of a senescence-associated secretory phenotype (SASP) (interleukin-6 and interleukin-8 (IL-6 and IL-8)). Additionally, we found that the inhibition of p65 prevents the transmission of paracrine senescence between mesenchymal stem cells and the proinflammatory message through small extracellular vesicles. Our work highlights the important role of p65 and its inhibition to restore the loss of functionality of small extracellular vesicles from senescent mesenchymal stem cells and their inflamm-aging signature.Instituto de Salud Carlos III; PI20/00497Xunta de Galicia; ED481B 2017/11

Therapy free of cells vs human mesenchymal stem cells from umbilical cord stroma to treat the inflammation in OA

[Abstract] Osteoarthritis (OA) is closely linked to the increase in the number of senescent cells in joint tissues, and the senescence-associated secretory phenotype (SASP) is implicated in cartilage degradation. In the last decade, extracellular vesicles (EV) in combination with the use of miRNAs to modify post-transcriptional expressions of multiple genes have shown their utility in new therapies to treat inflammatory diseases. This work delves into the anti-inflammatory effect of extracellular vesicles derived from mesenchymal stem cells (MSC) previously modified to inhibit the expression of miR-21. We compare the efficacy of two treatments, MSC with their miR-21 inhibited through lentiviral transfection and their EV, against inflammation in a new OA animal model. The modified MSC and their EV were intraperitoneally injected in an OA animal model twice. One month after treatment, we checked which therapy was the most effective to reduce inflammation compared with animals untreated. Treated OA model sera were analyzed for cytokines and chemokines. Subsequently, different organs were analyzed to validate the results obtained. EV were the most effective treatment to reduce chemokines and cytokines in serum of OA animals as well as SASP, in their organs checked by proteomic and genomic techniques, compared with MSC alone in a statistically significant way. In conclusion, MSC-miR-21--derived EV showed a higher therapeutic potential in comparison with MSCs-miR-21-. They ameliorate the systemic inflammation through inactivation of ERK1/2 pathway in OA in vivo model. Workflow of the realization of the animal model of OA by injecting cells into the joint cavity of the left knee of the animals, which produces an increase in serum cytokines and chemokines in the animals in addition to the increase in SASP and markers of inflammation. Inhibition of miR-21 in MSCs, from the stroma of the human umbilical cord, by lentivirus and extraction of their EVs by ultracentrifugation. Finally, application of MSC therapy with its miR-21 inhibited or its EVs produces a decrease in serum cytokines and chemokines in the treated animals, in addition to an increase in SASP and markers of inflammation. The cell-free therapy being the one that produces a greater decrease in the parameters studied.Xunta de Galicia; ED481D-2021-020Instituto de Salud Carlos III; PI20/0049

Action Mechanisms of Small Extracellular Vesicles in Inflammaging [Review]

This article belongs to the Special Issue Extracellular Vesicles Research in Inflamm-Aging[Abstract] The accumulation process of proinflammatory components in the body due to aging influences intercellular communication and is known as inflammaging. This biological mechanism relates the development of inflammation to the aging process. Recently, it has been reported that small extracellular vesicles (sEVs) are mediators in the transmission of paracrine senescence involved in inflammatory aging. For this reason, their components, as well as mechanisms of action of sEVs, are relevant to develop a new therapy called senodrugs (senolytics and senomorphic) that regulates the intercellular communication of inflammaging. In this review, we include the most recent and relevant studies on the role of sEVs in the inflammatory aging process and in age-related diseases such as cancer and type 2 diabetes.J.F.L. was funded by Xunta de Galicia, Grant Number ED481D-2021-020. M.C.A. received a grant from the Spanish National Health Institute Carlos III (PI20/00497)Xunta de Galicia; ED481D-2021-02

Shotgun proteomics reveals senomorphic targets based on SASP-mediated by small extracellular vesicles

Poster.-- Human Proteome Organization World Congress, HUPO 2023, 17-21 SeptemberCells have the capacity to modulate the microenvironment through the secretion of molecules (cytokines, chemokines, matrix proteins…) and vesicles, which vary according to the pathology involved, such as cancer or natural process such as senescence. Cellular senescence is a process that enhance with ageing and its association with the onset of age-related diseases. The process is characterized by less proliferation, increased β-galactosidase activity and specific secretory phenotype known as SASP. SASP lead the microenvironment to a more pro-inflammatory one and has the capacity to induce paracrine senescence in neighbouring cells. The regulation of small extracellular vesicles (sEV), which are part of SASP, have a high potential to develop drugs that modulate the senescence transmission. Our aim is to find a proteomic signature of the SASP mediated by sEV to reveal pathways associated with the senescence transmissionN

Study of Ferroptosis Transmission by Small Extracellular Vesicles in Epithelial Ovarian Cancer Cells

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. The current treatment for EOC involves surgical debulking of the tumors followed by a combination of chemotherapy. While most patients achieve complete remission, many EOCs will recur and develop chemo-resistance. The cancer cells can adapt to several stress stimuli, becoming resistant. Because of this, new ways to fight resistant cells during the disease are being studied. However, the clinical outcomes remain unsatisfactory. Recently, ferroptosis, a novel form of regulated cell death trigged by the accumulation of iron and toxic species of lipid metabolism in cells, has emerged as a promising anti-tumor strategy for EOC treatment. This process has a high potential to become a complementary treatment to the current anti-tumor strategies to eliminate resistant cells and to avoid relapse. Cancer cells, like other cells in the body, release small extracellular vesicles (sEV) that allow the transport of substances from the cells themselves to communicate with their environment. To achieve this, we analyzed the capacity of epithelial ovarian cancer cells (OVCA), treated with ferroptosis inducers, to generate sEV, assessing their size and number, and study the transmission of ferroptosis by sEV. Our results reveal that OVCA cells treated with ferroptotic inducers can modify intercellular communication by sEV, inducing cell death in recipient cells. Furthermore, these receptor cells are able to generate a greater amount of sEV, contributing to a much higher ferroptosis paracrine transmission. Thus, we discovered the importance of the sEV in the communication between cells in OVCA, focusing on the ferroptosis process. These findings could be the beginning form to study the molecular mechanism ferroptosis transmission through sEV