Metals Exposure and Cardiovascular Health: Characterizing Novel Risk Factors of Heart Failure

Heart Failure is a leading cause of death and disability worldwide. The identification of risk factors of heart failure in healthy individuals is key to improve disease prevention and reduce mortality. Metals exposures are recently established cardiovascular disease risk factors, but their association with heart failure remains understudied and prospective studies across diverse populations are needed. Metals are widespread in the environment, some of the sources of exposure include drinking water, air, and soil contamination. Some population groups, particularly American Indian, Hispanic/Latino, and Black communities in the United States are exposed to higher levels of environmental metals as a result of sociodemographic and structural factors including structural racism. These population groups suffer a higher burden of heart failure compared to White populations. Importantly, the burden of heart failure in American Indian communities in the United States, a population group with high rates of diabetes, hypertension, and other cardiovascular disease risk factors, is underreported, and key risk factors of heart failure in these population groups remain understudied. This dissertation characterized relevant risk factors of heart failure in American Indian participants from the Strong Heart Study. Towards the goal of identifying novel preventable cardiovascular disease risk factors, it comprehensively assessed the sources of exposure and biomarkers for multiple non-essential and essential metals with a focus on characterizing drivers of disparities in drinking water metal concentrations. Then, it evaluated the role of exposure to multiple metals (individually and as a mixture) on the risk of heart failure and overall cardiovascular disease and all-cause mortality, leveraging three geographically and racially and ethnically diverse population-based cohorts: the Multi-Ethnic Study of Atherosclerosis (MESA), the Strong Heart Study (SHS), and the Hortega cohort. Last, it identified and evaluated new opportunities for the mitigation of metal toxicity through nutritional interventions. Chapter 1 provides background information about heart failure epidemiology and pathophysiology, the role of environmental metals on cardiovascular disease, and introduces the dissertation framework necessary to contextualize the work included in this dissertation. Chapter 2 estimated the incidence of heart failure in the SHS, a large epidemiological cohort of American Indian adults from Arizona, Oklahoma, North Dakota, and South Dakota, followed from 1989-1991 through 2019. A parsimonious heart failure-risk prediction equation that accounts for relevant cardiovascular risk factors affecting American Indian communities was developed. The incidence rate of heart failure was 9.5 per 1,000 person-years, with higher rates across participants with diabetes, hypertension, and albuminuria. Significant predictors for heart failure risk at 5 and 10 years included age, smoking, albuminuria, and previous myocardial infarction. Diabetes diagnosis and higher levels of HbA1c were significant predictors of risk at 10 and 28 years. Models achieved a high discrimination performance (C-index (95%CI): 0.81 (0.76, 0.84) at 5 years, 0.78 (0.75, 0.81) at 10 years, and 0.77 (0.74, 0.78) up to 28 years), and some associations varied across HF subtypes. Chapter 3 developed a comprehensive overview of the main sources and routes of exposure, biotransformation, and biomarkers of exposure and internal dose for 12 metals/metalloids, including 8 non-essential elements (arsenic, barium, cadmium, lead, mercury, nickel, tin, uranium) and 4 essential elements (manganese, molybdenum, selenium, and zinc), providing a set of recommendations for the use and interpretation of metal biomarkers in epidemiological studies. Chapter 4 conducted the first nationwide geospatial analysis identifying racial/ethnic inequalities in arsenic and uranium concentrations in public drinking water across the conterminous United States using geospatial models. The association between county-level racial/ethnic composition and public water arsenic and uranium concentrations (2000-2011)was assessed. Higher proportions of Hispanic/Latino and American Indian/Alaskan Native residents were associated with 6% (95% CI: 4-8%), and 7% (3-11%) higher levels of arsenic, and 17% (13-22%), and 2% (-4-8%), higher levels of uranium, respectively, in public drinking water, after accounting for relevant social and geological indicators. Higher county-level proportions of non-Hispanic Black residents were associated with higher arsenic and uranium in the Southwest, where concentrations of these contaminants are high. These findings identified the key role of structural racism as driver of drinking water metal concentrations inequalities. Chapter 5 evaluated the prospective association between urinary metal levels, a established biomarker of internal dose, and incident heart failure across three geographically and ethnically/racially diverse cohorts: MESA and SHS in the United States, and the Hortega Study in Spain. These findings consistently identified significant associations across cohorts for cadmium (pooled hazard ratio: 1.15 (95% CI: 1.07, 1.24), tungsten (1.07 (1.02, 1.12)), copper (1.31 (1.18, 1.45)), molybdenum (1.13 (1.05, 1.22)), and zinc (1.22 (1.14, 1.32))). Higher levels of urinary metals analyzed as a mixture were significantly associated with increased incident heart failure risk in MESA and SHS, and non-significantly increased in the Hortega Study, which has a smaller number of events. Chapter 6 assessed the prospective association of urinary metals with incident cardiovascular disease and all-cause mortality in MESA, including a total of 6,599 participants at baseline (2000-2001), followed through 2019. Significant associations between higher levels of urinary cadmium, tungsten, uranium, cobalt, copper, and zinc, and higher risk of CVD and all-cause mortality were identified. A positive linear dose-response was identified for cadmium and copper with both endpoints. The adjusted HRs (95%CI) for an interquartile range (IQR) increase in the mixture of these six urinary metals and the correspondent 10-year survival probability difference (95% CI) were 1.29 (1.11, 1.56), and -1.1% (-2.0, -0.05) for incident CVD and 1.66 (1.47, 1.91), and -2.0% (-2.6, -1.5) for all-cause mortality. Chapter 7 investigated the effects of a nutritional intervention with folic acid (FA) and B12 supplementation on arsenic methylation in children exposed to high levels of drinking water arsenic in Bangladesh. The randomized controlled trial included a total of 240 children 8-11 years old. Compared to placebo, the supplementation group experienced a significant increase in the concentration of blood DMA, a non-toxic arsenic metabolite, by 14.0% (95%CI: 5.0, 25.0) and blood secondary methylation index (DMAs/MMAs) by 0.19 (95%CI: 0.09, 0.35). Similarly, there was a 1.62% (95%CI: 0.43, 2.83) significantly higher urinary %DMAs and -1.10% (CI: -1.73, -0.48) significantly lower urinary %MMAs compared to placebo group after 1 week. These results confirmed that FA+B12 supplementation increases arsenic methylation in children as reflected by decreased MMAs and increased DMAs in blood and urine. Altogether, the findings presented in this dissertation consistently identify the role of urinary metals as robust risk factors of heart failure, overall cardiovascular disease and all-cause mortality across diverse populations. With consistent findings across multiple assessments of the dose response relationship and mixture approaches. Additionally, this dissertation work contributes to address disparities in environmental exposures and heart failure burden, respectively, by characterizing the impact of structural racism drinking water metal exposures disparities and identifying relevant risk factors of heart failure in American Indian populations who are historically underrepresented in epidemiological cohorts. Last, this dissertation identifies the role of folic acid and B12 supplementation to reduce arsenic toxicity in children. These findings have direct clinical and policy implications, as they can inform the development of novel clinical guidelines to incorporate environmental factors in clinical risk prediction, and they can inform drinking water regulation and infrastructure efforts to support at risk communities and inform population-level nutritional recommendations and policies

Fabrication of 3D printed Gelatin-Hyaluronic Acid hydrogels based on enzyme-mediated tyramine conjugates and other derivatives

Extracellular matrices (ECMs) in soft tissues are highly hydrated structures mainly composed of glycoproteins (such as collagen or fibronectin) and glycosaminoglycans (such as hyaluronic acid (HA) or keratan sulfate), each one with a tissue-specific composition (1). Many of these tissues are unable to regenerate themselves or can only repair minor injuries, as is the case of skin (2), heart (3) and cartilage (4). Hydrogels are hydrophilic polymeric networks with high water retention capability which have been frequently proposed as potential candidates for soft tissues regeneration due to their tunable physical, chemical and biological properties, biocompatibility and their ability to mimic the native ECMs (5-7). Besides, they promote phenotype maintenance and induce re-differentiation of different cells such as cardiomyocytes (8), chondrocytes (9) and hepatoblast (10). Most hydrogels need to be chemically cross-linked to not dissolve at body temperature. Conventional cross-linking methods involving chemical reactions are generally cytotoxic. Solvents, initiators or unreacted substances are left behind, often resulting in inflammation and cell death (11-13). In order to prevent any harmful effect on cells, they must be therefore pre-formed under safe lab conditions, thoroughly washed and sterilised before implantation. Cross-linking reactions mediated by enzymes (14-16), also known as enzyme-mediated or enzyme-catalysed cross-linking, have been proposed relatively recently as a less problematic alternative for hydrogel scaffolding. In these systems, aqueous hydrogel precursor solutions are mixed with cellular components and/or desired bioactive agents prior to injection into the defect area. Enzymes, included or subsequently added to the precursor solutions, catalyse the cross-linking reaction immediately upon injection, generating covalent bonds between specific functional groups found within the polymer side chains. These mild in situ reactions, which can take place in a matter of seconds or minutes, do not produce any cytotoxic effects (17,18) and present several advantages (19-21) over pre-created hydrogels: adaptation to the shape of the defect, lower risk of implant migration, easy and effective cell encapsulation and deliverability, and minimally invasive surgical interventions that improve patient compliance and recovery (18,22-27). Gelatin (Gel) is a natural polymer derived from the partial denaturation of collagen that has attracted attention as a hydrogel scaffold into which cells can be embedded. It has accessible functional groups that can react with other molecules and different integrin-binding sites for cell adhesion and differentiation (28). However, its poor mechanical properties limit its applications. This lack of mechanical strength can be overcome by preparing blends of gelatin with other polymers (29) by enzyme-mediated reactions such as hyaluronic acid (HA). HA is well known for its high hydrophilicity, good lubrication, biocompatibility, and low cell and protein adhesive properties (30). Gel-HA hydrogels enzymatically cross-linked by horseradish peroxidase (HRP) and hydrogen peroxide (H202) through the covalent bonding of tyramine (Tyr) have demonstrated their non-cytotoxicity and potential for cell adhesion and spreading (33-35). HA concentration in this system can be modified according to the required stiffness, water sorption, pore size and gelation time (18, 31, 32), which gives rise to potential candidates for several types of soft tissue models, regeneration strategies and applications in minimally invasive procedures. Traditional approaches based on hydrogel or other soft materials for scaffolding are limited in their capacity of producing complex microstructures with accurate biomimetic properties. Three-dimensional (3D) bioprinting technologies to the contrary, offer a novel versatility to co-deliver cells and biomaterials with precise control over their configurations, spatial distributions and pattern exactitude, achieving personalized constructs that mimic the functionality of target tissues and organs (22, 36-42). One of the most appealing applications of 3D bioprinting nowadays is the development of functional 3D tissue models. Current 2D cell cultures, particularly the animal models employed for in vitro drug testing, are shown to respond differently to drug candidates compared to humans, and hence their use as models of human diseases or medical conditions result ineffective and futile (163). However, like any other new and complex technology, the process towards its complete implementation still has a long way to go. The determination and understanding of the parameters involved in a process of hydrogel printing as well as the effects of their combination are paramount for the success of the scaffold and can present a challenge even to the most veteran researchers. In this work, we propose a viable and reproducible cell encapsulation protocol of Gel-Tyr/HA-Tyr hydrogels by means of 3D bioprinting for in vitro drug testing and further use in regenerative medicine, significantly reducing the worker’s laboratory time and facilitating the completion of long laborious tasks in multi-sample hydrogel generation. We also provide an extensive and well-documented description of several parameters directly involved in every process of printing design and protocol optimisation as well as some of their common individual effects on printed scaffolds

Radio interferometric observations of candidate water-maser-emitting planetary nebulae

We present Very Large Array (VLA) observations of H2O and OH masers, as well as radio continuum emission at 1.3 and 18 cm toward three sources previously cataloged as planetary nebulae (PNe) and in which single-dish detections of H2O masers have been reported: IRAS 17443-2949, IRAS 17580-3111, and IRAS 18061-2505. Our goal was to unambiguously confirm their nature as water-maser-emitting PNe, a class of objects of which only two bona-fide members were previously known. We detected and mapped H2O maser emission toward all three sources, while OH maser emission is detected in IRAS 17443-2949 and IRAS 17580-3111 as well as in other two objects within the observed fields: IRAS 17442-2942 (unknown nature) and IRAS 17579-3121 (also cataloged as a possible PN). We found radio continuum emission associated only with IRAS 18061-2505. Our results confirm IRAS 18061-2505 as the third known case of a PN associated with H2O maser emission. The three known water-maser-emitting PNe have clear bipolar morphologies, which suggests that water maser emission in these objects is related to non-spherical mass-loss episodes. We speculate that these bipolar PNe would have ``water-fountain'' Asymptotic Giant Branch (AGB) and post-AGB stars as their precursors. A note of caution is given for other objects that have been classified as OHPNe (objects with both OH maser and radio continuum emission, that could be extremely young PNe) based on single-dish observations, since interferometric data of both OH masers and continuum are necessary for a proper identification as members of this class.Comment: 33 pages, 10 figures. Accepted by The Astronomical Journa

Por la illustrissima señora condesa de Morata. En la revocacion de la firma del illustrissimo Conde de Sastago

Copia digital : Diputación Provincial de Zaragoza. Servicio de Archivos y Bibliotecas, 2010En otro documento del mismo proceso se da la fecha de 163

Urinary Metal Levels and Coronary Artery Calcification: Longitudinal Evidence in the Multi-Ethnic Study of Atherosclerosis (MESA)

Objective: Growing evidence indicates that exposure to metals are risk factors for cardiovascular disease (CVD). We hypothesized that higher urinary levels of metals with prior evidence of an association with CVD, including non-essential (cadmium , tungsten, and uranium) and essential (cobalt, copper, and zinc) metals are associated with baseline and rate of change of coronary artery calcium (CAC) progression, a subclinical marker of atherosclerotic CVD. Methods: We analyzed data from 6,418 participants in the Multi-Ethnic Study of Atherosclerosis (MESA) with spot urinary metal levels at baseline (2000-2002) and 1-4 repeated measures of spatially weighted coronary calcium score (SWCS) over a ten-year period. SWCS is a unitless measure of CAC highly correlated to the Agatston score but with numerical values assigned to individuals with Agatston score=0. We used linear mixed effect models to assess the association of baseline urinary metal levels with baseline SWCS, annual change in SWCS, and SWCS over ten years of follow-up. Urinary metals (adjusted to μg/g creatinine) and SWCS were log transformed. Models were progressively adjusted for baseline sociodemographic factors, estimated glomerular filtration rate, lifestyle factors, and clinical factors. Results: At baseline, the median and interquartile range (25th, 75th) of SWCS was 6.3 (0.7, 58.2). For urinary cadmium, the fully adjusted geometric mean ratio (GMR) (95%Cl) of SWCS comparing the highest to the lowest quartile was 1.51 (1.32, 1.74) at baseline and 1.75 (1.47, 2.07) at ten years of follow-up. For urinary tungsten, uranium, and cobalt the corresponding GMRs at ten years of follow-up were 1.45 (1.23, 1.71), 1.39 (1.17, 1.64), and 1.47 (1.25, 1.74), respectively. For copper and zinc, the association was attenuated with adjustment for clinical risk factors; GMRs at ten years of follow-up before and after adjustment for clinical risk factors were 1.55 (1.30, 1.84) and 1.33 (1.12, 1.58), respectively, for copper and 1.85 (1.56, 2.19) and 1.57 (1.33, 1.85) for zinc. Conclusion: Higher levels of cadmium, tungsten, uranium, cobalt, copper, and zinc, as measured in urine, were associated with subclinical CVD at baseline and at follow-up. These findings support the hypothesis that metals are pro-atherogenic factors.The Multi-Ethnic Study of Atherosclerosis (MESA) is supported by contracts 75N92020D00001, HHSN268201500003I, N01-HC-95159, 75N92020D00005, N01-HC-95160, 75N92020D00002, N01-HC-95161, 75N92020D00003, N01-HC-95162, 75N92020D00006, N01-HC-95163, 75N92020D00004, N01-HC-95164, 75N92020D00007, N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168 and N01-HC-95169 from the National Heart, Lung, and Blood Institute, and by grants UL1-TR-000040, UL1-TR-001079, and UL1-TR-001420 from the National Center for Advancing Translational Sciences (NCATS). This publication was developed under the Science to Achieve Results (STAR) research assistance agreements, No. RD831697 (MESA Air) and RD-83830001 (MESA Air Next Stage), awarded by the U.S Environmental Protection Agency (EPA). It has not been formally reviewed by the EPA. The views expressed in this document are solely those of the authors and the EPA does not endorse any products or commercial services mentioned in this publication. Dr. Maria Tellez-Plaza was supported by grants PI15/00071 and PI22/00029 from the Strategic Action for Health Research, Instituto de Salud Carlos III and the Spanish Ministry of Science and Innovation, and co-funded with European Funds for Regional Development (FEDER). The opinions and views expressed in this article are those of the authors and do not necessarily represent the official position of the Instituto de Salud Carlos III (Spain). Work in the authors? laboratories is also supported in part by NIH grants P42ES023716, P42ES010349, P42ES033719, P30ES009089, T32ES007322, R01ES029967, R01HL155576. The authors thank the other investigators, the staff, and the participants of the MESA study for their valuable contributions. A full list of participating MESA investigators and institutions can be found at http://www.mesa-nhlbi.org. This paper has been reviewed and approved by the MESA Publications and Presentations Committee.N

“Energetic” cancer stem cells (e-CSCs) : a new hyper-metabolic and proliferative tumor cell phenotype, driven by mitochondrial energy

Here, we provide the necessary evidence that mitochondrial metabolism drives the anchorage-independent proliferation of CSCs. Two human breast cancer cell lines, MCF7 [ER(+)] and MDA-MB-468 (triple-negative), were used as model systems. To directly address the issue of metabolic heterogeneity in cancer, we purified a new distinct sub-population of CSCs, based solely on their energetic profile. We propose the term “energetic” cancer stem cells (e-CSCs), to better describe this novel cellular phenotype. In a single step, we first isolated an auto-fluorescent cell sub-population, based on their high flavin-content, using flow-cytometry. Then, these cells were further subjected to a detailed phenotypic characterization.More specifically, e-CSCs weremore glycolytic, with higher mitochondrial mass and showed significantly elevated oxidative metabolism. e-CSCs also demonstrated an increased capacity to undergo cell cycle progression, as well as enhanced anchorage-independent growth and ALDH-positivity. Most importantly, these e-CSCs could be effectively targeted by treatments with either (i) OXPHOS inhibitors (DPI) or (ii) a CDK4/6 inhibitor (Ribociclib). Finally, we were able to distinguish two distinct phenotypic sub-types of e-CSCs, depending on whether they were grown as 2D-monolayers or as 3D-spheroids. Remarkably, under 3D anchorage-independent growth conditions, e-CSCs were strictly dependent on oxidative mitochondrial metabolism. Unbiased proteomics analysis demonstrated the up-regulation of gene products specifically related to the anti-oxidant response, mitochondrial energy production, and mitochondrial biogenesis. Therefore,mitochondrial inhibitors should be further developed as promising anti-cancer agents, to directly target and eliminate the “fittest” e-CSCs. Our results have important implications for using e-CSCs, especially those derived from 3D-spheroids, (i) in tumor tissue bio-banking and (ii) as a new cellular platform for drug development

D2.4 - Final Bundle of Client-side Components

This document describes the final bundle of client-side components, including descriptions of their functionality, and links to their full designs and downloadable versions. This bundle aggregates only the WP2 assets. Other client-side assets not covered here will be addressed in the final WP3 deliverables. Those assets created and licenced as open software will be continuously improved and maintained by their creators until the end of the project (the task has been extended to month 48) and beyond. For a full description of the related server-side components, please refer to D2.2 - Final Bundle of Server-side Components.This study is part of the RAGE project. The RAGE project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 644187. This publication reflects only the author's view. The European Commission is not responsible for any use that may be made of the information it contains