Endothelial Senescence and the Chronic Vascular Diseases: Challenges and Therapeutic Opportunities in Atherosclerosis

Atherosclerosis is probably one of the paradigms of disease linked to aging. Underlying the physiopathology of atherosclerosis are cellular senescence, oxidative stress, and inflammation. These factors are increased in the elderly and from chronic disease patients. Elevated levels of oxidative stress affect cellular function and metabolism, inducing senescence. This senescence modifies the cell phenotype into a senescent secretory phenotype. This phenotype activates immune cells, leading to chronic systemic inflammation. Moreover, due to their secretory phenotype, senescence cells present an increased release of highlighted extracellular vesicles that will change nearby/neighborhood cells and paracrine signaling. For this reason, searching for specific senescent cell biomarkers and therapies against the development/killing of senescent cells has become relevant. Recently, senomorphic and senolityc drugs have become relevant in slowing down or eliminating senescence cells. However, even though they have shown promising results in experimental studies, their clinical use is still yet to be determinedInstituto de Salud Carlos IIISociedad Española de NefrologíaComunidad de MadridFondo Europeo de Desarrollo Regiona

Molecular and Structural Discrimination of Proline Racemase and Hydroxyproline-2-Epimerase from Nosocomial and Bacterial Pathogens

The first eukaryotic proline racemase (PRAC), isolated from the human Trypanosoma cruzi pathogen, is a validated therapeutic target against Chagas' disease. This essential enzyme is implicated in parasite life cycle and infectivity and its ability to trigger host B-cell nonspecific hypergammaglobulinemia contributes to parasite evasion and persistence. Using previously identified PRAC signatures and data mining we present the identification and characterization of a novel PRAC and five hydroxyproline epimerases (HyPRE) from pathogenic bacteria. Single-mutation of key HyPRE catalytic cysteine abrogates enzymatic activity supporting the presence of two reaction centers per homodimer. Furthermore, evidences are provided that Brucella abortus PrpA [for ‘proline racemase’ virulence factor A] and homologous proteins from two Brucella spp are bona fide HyPREs and not ‘one way’ directional PRACs as described elsewhere. Although the mechanisms of aminoacid racemization and epimerization are conserved between PRAC and HyPRE, our studies demonstrate that substrate accessibility and specificity partly rely on contraints imposed by aromatic or aliphatic residues distinctively belonging to the catalytic pockets. Analysis of PRAC and HyPRE sequences along with reaction center structural data disclose additional valuable elements for in silico discrimination of the enzymes. Furthermore, similarly to PRAC, the lymphocyte mitogenicity displayed by HyPREs is discussed in the context of bacterial metabolism and pathogenesis. Considering tissue specificity and tropism of infectious pathogens, it would not be surprising if upon infection PRAC and HyPRE play important roles in the regulation of the intracellular and extracellular amino acid pool profiting the microrganism with precursors and enzymatic pathways of the host

A conserved structure within the HIV gag open reading frame that controls translation initiation directly recruits the 40S subunit and eIF3

Translation initiation on HIV genomic RNA relies on both cap and Internal Ribosome Entry Site (IRES) dependant mechanisms that are regulated throughout the cell cycle. During a unique phenomenon, the virus recruits initiation complexes through RNA structures located within Gag coding sequence, downstream of the initiation codon. We analyzed initiation complexes paused on the HIV-2 gag IRES and revealed that they contain all the canonical initiation factors except eIF4E and eIF1. We report that eIF3 and the small ribosomal subunit bind HIV RNA within gag open reading frame. We thus propose a novel two step model whereby the initial event is the formation of a ternary eIF3/40S/IRES complex. In a second step, dependent on most of the canonical initiation factors, the complex is rearranged to transfer the ribosome on the initiation codons. The absolute requirement of this large structure for HIV translation defines a new function for a coding region. Moreover, the level of information compaction within this viral genome reveals an additional level of evolutionary constraint on the coding sequence. The conservation of this IRES and its properties in rapidly evolving viruses suggest an important role in the virus life cycle and highlight an attractive new therapeutic target

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

A high magnesium concentration in citrate dialysate prevents oxidative stress and damage in human monocytes in vitro

Background. The use of dialysis fluids (DFs) during haemodialysis has been associated with increased oxidative stress and reduced serum magnesium (Mg) levels, contributing to chronic inflammation. Since the role of Mg in modulating immune function and reducing oxidative stress has been demonstrated, the aim of this study was to characterize in vitro whether increasing the Mg concentration in DFs could protect immune cells from oxidative stress and damage. Methods. The effect of citrate [citrate dialysis fluid (CDF), 1 mM] or acetate [acetate dialysis fluid (ADF), 3 mM] dialysates with low (0.5 mM; routinely used) or high (1 mM, 1.25 mM and 2 mM) Mg concentrations was assessed in THP-1 human monocytes. The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and oxidized/reduced (GSSG/GSH) glutathione were quantified under basal and inflammatory conditions (stimulation with lipopolysaccharide, LPS). Results. The increase of Mg in CDF resulted in a significant reduction of ROS production under basal and inflammatory conditions (extremely marked in 2 mM Mg; P< 0.001). These effects were not observed in ADF. Interestingly, in a dose-dependent manner, high Mg doses in CDF reduced oxidative stress in monocytes under both basal and inflammatory conditions. In fact, 2 mM Mg significantly decreased the levels of GSH, GSSG and MDA and the GSSG/GSH ratio in relation to 0.5 mM Mg. Conclusions. CDF produces lower oxidative stress than ADF. The increase of Mg content in DFs, especially in CDF, could have a positive and protective effect in reducing oxidative stress and damage in immune cells, especially under inflammatory conditions.Instituto de Salud Carlos IIISociedad Española de NefrologíaUniversidad Complutense de MadridUniversidad de AlcaláFondo Europeo de Desarrollo Regional-FEDE

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

Distinct roles for the IIId2 sub-domain in pestivirus and picornavirus internal ribosome entry sites.

Viral internal ribosomes entry site (IRES) elements coordinate the recruitment of the host translation machinery to direct the initiation of viral protein synthesis. Within hepatitis C virus (HCV)-like IRES elements, the sub-domain IIId(1) is crucial for recruiting the 40S ribosomal subunit. However, some HCV-like IRES elements possess an additional sub-domain, termed IIId2, whose function remains unclear. Herein, we show that IIId2 sub-domains from divergent viruses have different functions. The IIId2 sub-domain present in Seneca valley virus (SVV), a picornavirus, is dispensable for IRES activity, while the IIId2 sub-domains of two pestiviruses, classical swine fever virus (CSFV) and border disease virus (BDV), are required for 80S ribosomes assembly and IRES activity. Unlike in SVV, the deletion of IIId2 from the CSFV and BDV IRES elements impairs initiation of translation by inhibiting the assembly of 80S ribosomes. Consequently, this negatively affects the replication of CSFV and BDV. Finally, we show that the SVV IIId2 sub-domain is required for efficient viral RNA synthesis and growth of SVV, but not for IRES function. This study sheds light on the molecular evolution of viruses by clearly demonstrating that conserved RNA structures, within distantly related RNA viruses, have acquired different roles in the virus life cycles

Papel de las proteínas oxidadas sobre el daño endotelial asociado a la enfermedad renal crónica : aproximación “in vivo” e “in vitro”

Una manifestación temprana de las enfermedades cardiovasculares es la disfunción endotelial, asociada a un entorno inflamatorio propio de patologías crónicas como la insuficiencia renal crónica (IRC). Está descrito que en la IRC se producen modificaciones pos-traduccionales en las proteínas, tales como la oxidación, que serían capaces de generar un daño endotelial y como consecuencia producir eventos cardiovasculares asociados a esta patología (IRC). Por ello, nos centramos en caracterizar y determinar el papel de estas proteínas oxidadas presentes en los plasmas de pacientes con IRC. Además, valoramos los efectos de una proteína en concreto, albúmina humana, sobre cultivos endoteliales, para relacionar estas moléculas alteradas presentes en plasmas de sujetos patológicos y nuestra proteína modificada en cuanto a la capacidad de provocar fenómenos de daño y senescencia prematura inducida en el endotelio, con las consiguientes complicaciones fisiopatológicas. Se realizó una aproximación in vivo, en la cual, mediante técnica ELISA, se determinó la cantidad (ng/mL) de AOPP (Advance Oxidized Protein Products) en plasmas de sujetos (n=18) con IRC, procedentes del Hospital Puerta de Hierro, Majadahonda. Paralelamente, se cuantificó el número de micropartículas (MPs) endoteliales en las muestras para observar el daño endotelial.”In Vitro”, se utilizó Albúmina Humana Oxidada (mediante sulfato de cobre) como tratamiento, para ver los posibles efectos deletéreos de la misma, medidos como senescencia y daño (actividad B-gal, producción de MPs, reendotelización y proliferación). Se observó una mayor cantidad de proteínas oxidadas así como mayor número de MPs endoteliales en los plasmas de los sujetos respecto el control; se valoró que la presencia de albúmina oxidada induce senescencia prematura en el endotelio, así como expresión de mayor número de MPs e inhibición parcial de la proliferación y reendotelización del mismo, además de una mayor expresión de ciclina D1 a las 6 horas de tratamiento, proteína relacionada con la senescenci

Molino de nueva invención, construido de orden y por cuenta del Rey ... en el Real Posito de Madrid

Cédula 17720408Sign.: A8Portada y texto con orla tip.La h. de lám. es grabado calcográfico del molino: "Tejada del.