Endothelial Cell Senescence in the Pathogenesis of Endothelial Dysfunction

Aging is the main risk factor for cardiovascular diseases (CVD), and senescence in endothelial cells seems to be an initial step in the cascade of events that will culminate with the development of these pathologies. In this chapter, we examine the pathophysiological mechanism(s) involved in endothelial senescence, leading to CVD as well as the biochemical and cellular pathways that may explain the activation and development of the process of endothelial senescence, and we discuss new hypotheses supported by experimental results which suggest that the senescent endothelial cell may induce a general process of vascular senescence. This process is probably induced either by soluble molecules secreted by these senescent cells and/or by intercellular signals transported in cellular vesicles that may be useful as biomarkers and as potential therapeutic targets in endothelial senescence

Papel de las microvesículas como biomarcadores y futuras dianas farmacológicas de enfermedades cardiovasculares

In the 1990´s, it was discovered a new cell-cell communication system based on the action of vesicles that cargo bioactive molecules, on neighboring cells. These vesicles, known as extracellular vesicles (EVs) act as regulators of several physiological processes but also participate in the development and progression of multiple diseases. Microvesicles (MVs) are types of EVs that are implicated in the etiopathogenesis of a large number of cardiovascular diseases due to they take part in the onset of atherosclerosis. Different cardioprotective drugs have shown to have an effect on MVs. In addition, since the discovery that MVs are capable of transferring biological information, the use of them as drug delivery vehicles has gained scientific interest. The aim of this work is to analyze the involvement of MVs in the origin of atherosclerosis to demonstrate their role as diagnostic biomarkers, as well as to review the pharmacological effect of current therapies on MVs and their role as therapeutic tool.En la década de los 90 se descubrió un nuevo sistema de comunicación célula-célula a través de vesículas con moléculas bioactivas liberadas al espacio extracelular. Estas vesículas, conocidas como vesículas extracelulares (VE), actúan como reguladores de procesos fisiológicos, pero también participan en el desarrollo y progresión de múltiples patologías. Las microvesículas (MVs) son un tipo de VE que se producen como resultado del daño celular, y están implicadas en la etiopatogenia de un gran número de enfermedades cardiovasculares porque intervienen en el inicio de la aterosclerosis. Diferentes fármacos cardioprotectores han demostrado tener un efecto sobre las MVs; por otro lado, desde que se conoce su capacidad para transferir información biológica, el uso de las éstas como vehículos de suministro molecular ha adquirido interés científico. El objetivo de este trabajo es analizar la implicación de las MVs en la etiopatogenia de la aterosclerosis estableciendo su importancia como biomarcadores de diagnóstico y de seguimiento. Se revisará el efecto farmacológico de las terapias actuales sobre las MVs y se discutirá su papel como herramienta terapéutica

Hypoxia-Inducible Factor-1α: The Master Regulator of Endothelial Cell Senescence in Vascular Aging

Aging is one of the hottest topics in biomedical research. Advances in research and medicine have helped to preserve human health, leading to an extension of life expectancy. However, the extension of life is an irreversible process that is accompanied by the development of aging-related conditions such as weakness, slower metabolism, and stiffness of vessels. It also debated that aging can be considered an actual disease with aging-derived comorbidities, including cancer or cardiovascular disease. Currently, cardiovascular disorders, including atherosclerosis, are considered as premature aging and represent the first causes of death in developed countries, accounting for 31% of annual deaths globally. Emerging evidence has identified hypoxia-inducible factor-1α as a critical transcription factor with an essential role in aging-related pathology, in particular, regulating cellular senescence associated with cardiovascular aging. In this review, we will focus on the regulation of senescence mediated by hypoxia-inducible factor-1α in age-related pathologies, with particular emphasis on the crosstalk between endothelial and vascular cells in age-associated atherosclerotic lesions. More specifically, we will focus on the characteristics and mechanisms by which cells within the vascular wall, including endothelial and vascular cells, achieve a senescent phenotyp

Indoxil sulfato induce senescencia y liberación de microvesículas en células endoteliales que promueven la calcificación vascular

Actas del V Congreso de Señalización Celular, SECUAH 2020. 16-18 de marzo, 2020. Universidad de Alcalá. Alcalá de Henares, Madrid. España.Antecedentes: Tanto los ancianos como los pacientes con enfermedad renal crónica (ERC) tienen una mayor probabilidad de sufrir enfermedades inflamatorias cardiovasculares (EICVs), las cuales pueden asociarse a la calcificación vascular como inicio de las mismas. Las células endoteliales tienen una senescencia prematura como mecanismo fisiopatológico en la ECV asociada a ERC. Hemos demostrado que las microvesículas (MVs) producidas por estas células senescentes, debido a su capacidad de comunicación intercelular, pueden iniciar dicha calcificación. Además, las toxinas urémicas, como el indoxil sulfato (IS), que se relacionan con la ERC, también pueden generar una senescencia endotelial precoz, lo que puede secretar MVs que inicien la calcificación vascular. Todo esto permite que las MVs puedan ser útiles para su caracterización como biomarcadores tempranos y/o diana terapéutica en estas patologías. Materiales y métodos: Se trataron células endoteliales (HUVEC) con IS. La senescencia se cuantificó por el método de β-galactosidasa. Por citometría de flujo se aislaron y se caracterizaron las MVs producidas por las HUVEC senescentes, y se utilizaron para el tratamiento de células musculares lisas (HASMC) durante 9, 20 y 30 días. En HASMC se valoraron los depósitos de calcio por el colorante rojo alizarina. A los 30 días, mediante el kit de fenolsufoneftaleina, se cuantificó el contenido de calcio. Mediante PCR cuantitativa se determinó en HASMC la expresión de genes pro-calcificantes y por western blot la expresión de un marcador específico de HASMC (SM22α). Resultados: El tratamiento con IS indujo senescencia precoz en HUVEC (control: 7,07±2,87% vs. IS: 72,90±13,09%, p<0.0001) y se observó un incremento en la producción de MVs (control: 1,68±1.00 vs. IS: 9.92±4,29 MVs/célula, p<0.005). Las MVs procedentes de células con senescencia precoz por el efecto del IS indujeron calcificación en HASMC, en un proceso asociado a desregulación en la expresión de genes procalcificantes (Runx2, BMP2). Además, las MVs generadas en HUVEC tratadas con IS producen un proceso de desdiferenciación de las HASMC (expresión de SM22α). Conclusión: Las células endoteliales que tienen una senescencia precoz como consecuencia del estrés oxidativo generado por las toxinas urémicas son capaces de producir MVs que promuevan la calcificación vascular. Su cuantificación en plasma permite que se pueda utilizar como biomarcador para valorar la progresión de la enfermedad en sujetos susceptibles y para diseñar dianas terapéuticas que eviten la generación o la presencia en el espacio extracelular de MVs procalcificantes

MicroRNA-126 regulates HypoxiaInducible Factor-1α which inhibited migration, proliferation, and angiogenesis in replicative endothelial senescence

Whereas a healthy endothelium maintains physiological vascular functions, endothelial damage contributes to the development of cardiovascular diseases. Endothelial senescence is the main determinant of endothelial dysfunction and thus of age-related cardiovascular disease. The objective of this study is to test the involvement of microRNA-126 and HIF-1α in a model of replicative endothelial senescence and the interrelationship between both molecules in this in vitro model. We demonstrated that senescent endothelial cells experience impaired tube formation and delayed wound healing. Senescent endothelial cells failed to express HIF-1α, and the microvesicles released by these cells failed to carry HIF-1α. Of note, HIF-1α protein levels were restored in HIF-1α stabilizer-treated senescent endothelial cells. Finally, we show that microRNA-126 was downregulated in senescent endothelial cells and microvesicles. With regard to the interplay between microRNA-126 and HIF-1α, transfection with a microRNA-126 inhibitor downregulated HIF-1α expression in early passage endothelial cells. Moreover, while HIF-1α inhibition reduced tube formation and wound healing closure, microRNA-126 levels remained unchanged. These data indicate that HIF-1α is a target of miRNA-126 in protective and reparative functions, and suggest that their therapeutic modulation could benefit age-related vascular disease

Bisphenol A Induces Accelerated Cell Aging in Murine Endothelium.

Bisphenol A (BPA) is a widespread endocrine disruptor affecting many organs and systems. Previous work in our laboratory demonstrated that BPA could induce death due to necroptosis in murine aortic endothelial cells (MAECs). This work aims to evaluate the possible involvement of BPA-induced senescence mechanisms in endothelial cells. The β-Gal assays showed interesting differences in cell senescence at relatively low doses (100 nM and 5 µM). Western blots confirmed that proteins involved in senescence mechanisms, p16 and p21, were overexpressed in the presence of BPA. In addition, the UPR (unfolding protein response) system, which is part of the senescent phenotype, was also explored by Western blot and qPCR, confirming the involvement of the PERK-ATF4-CHOP pathway (related to pathological processes). The endothelium of mice treated with BPA showed an evident increase in the expression of the proteins p16, p21, and CHOP, confirming the results observed in cells. Our results demonstrate that oxidative stress induced by BPA leads to UPR activation and senescence since pretreatment with N-acetylcysteine (NAC) in BPA-treated cells reduced the percentage of senescent cells prevented the overexpression of proteins related to BPA-induced senescence and reduced the activation of the UPR system. The results suggest that BPA participates actively in accelerated cell aging mechanisms, affecting the vascular endothelium and promoting cardiovascular diseases.post-print3206 K

Increasing the Magnesium Concentration in Various Dialysate Solutions Differentially Modulates Oxidative Stress in a Human Monocyte Cell Line

Oxidative stress is exacerbated in hemodialysis patients by several factors, including the uremic environment and the use of dialysis fluids (DFs). Since magnesium (Mg) plays a key role in modulating immune function and in reducing oxidative stress, we aimed to evaluate whether increasing the Mg concentration in different DFs could protect against oxidative stress in immunocompetent cells in vitro. Effect of ADF (acetate 3 mM), CDF (citrate 1 mM), and ACDF (citrate 0.8 mM + acetate 0.3 mM) dialysates with Mg at standard (0.5 mM) or higher (1, 1.25, and 2 mM) concentrations were assessed in THP-1 monocyte cultures. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels were quantified under basal and uremic conditions (indoxyl sulfate (IS) treatment). Under uremic conditions, the three DFs with 0.5 mM Mg promoted higher ROS production and lipid damage than the control solution. However, CDF and ACDF induced lower levels of ROS and MDA, compared to that induced by ADF. High Mg concentration (1.25 and/or 2 mM) in CDF and ACDF protected against oxidative stress, indicated by reduced ROS and MDA levels compared to respective DFs with standard concentration of Mg. Increasing Mg concentrations in ADF promoted high ROS production and MDA content. Thus, an increase in Mg content in DFs has differential effects on the oxidative stress in IS-treated THP-1 cells depending on the dialysate used.Instituto de Salud Carlos IIISociedad Española de Nefrologí

Targeting TLR4 with ApTOLL Improves Heart Function in Response to Coronary Ischemia Reperfusion in Pigs Undergoing Acute Myocardial Infarction.

Toll-like receptor 4 (TLR4) contributes to the pathogenesis of coronary ischemia/reperfusion (IR). To test whether the new TLR4 antagonist, ApTOLL, may prevent coronary IR damage, we administered 0.078 mg/kg ApTOLL or Placebo in pigs subjected to IR, analyzing the levels of cardiac troponins, matrix metalloproteinases, pro-, and anti-inflammatory cytokines, heart function, and tissue integrity over a period of 7 days after IR. Our results show that ApTOLL reduced cardiac troponin-1 24 h after administration, improving heart function, as detected by a significant recovery of the left ventricle ejection fraction (LVEF) and the shortening fraction (FS) cardiac parameters. The extension of necrotic and fibrotic areas was also reduced, as detected by Evans blue/2,3,5-triphenyltetrazolium chloride (TTC) staining, Hematoxylin/Eosine, and Masson Trichrome staining of heart sections, together with a significant reduction in the expression of the extracellular matrix-degrading, matrix metalloproteinase 9. Finally, the expression of the following cytokines, CCL1, CCL2, MIP1-A-B, CCL5, CD40L, C5/C5A, CXCL1, CXCL10, CXCL11, CXCL12, G-CSF, GM-CSF, ICAM-1, INF-g, IL1-a, ILI-b, IL-1Ra, IL2, IL4, IL5, IL6, IL8, IL10, IL12, IL13, IL16, IL17-A, IL17- E, IL18, IL21, IL27, IL32, MIF, SERPIN-E1, TNF-a, and TREM-1, were also assayed, detecting a pronounced decrease of pro-inflammatory cytokines after 7 days of treatment with ApTOLL. Altogether, our results show that ApTOLL is a promising new tool for the treatment of acute myocardial infarction (AMI).post-print3782 K

Mechanisms of cardiovascular disorders in patients with chronic kidney disease: a process related to accelerated senescence

Cardiovascular diseases (CVDs), especially those involving a systemic inflammatory process such as atherosclerosis, remain the leading cause of morbidity and mortality in patients with chronic kidney disease (CKD). CKD is a systemic condition affecting approximately 10% of the general population. The prevalence of CKD has increased over the past decades because of the aging of the population worldwide. Indeed, CVDs in patients with CKD constitute a premature form of CVD observed in the general population. Multiple studies indicate that patients with renal disease undergo accelerated aging, which precipitates the appearance of pathologies, including CVDs, usually associated with advanced age. In this review, we discuss several aspects that characterize CKD-associated CVDs, such as etiopathogenic elements that CKD patients share with the general population, changes in the cellular balance of reactive oxygen species (ROS), and the associated process of cellular senescence. Uremiaassociated aging is linked with numerous changes at the cellular and molecular level. These changes are similar to those observed in the normal process of physiologic aging. We also discuss new perspectives in the study of CKD-associated CVDs and epigenetic alterations in intercellular signaling, mediated by microRNAs and/or extracellular vesicles (EVs), which promote vascular damage and subsequent development of CVD. Understanding the processes and factors involved in accelerated senescence and other abnormal intercellular signaling will identify new therapeutic targets and lead to improved methods of diagnosis and monitoring for patients with CKD-associated CVDs