Aging Impairs Reverse Remodeling and Recovery of Ventricular Function after Isoproterenol-Induced Cardiomyopathy

Abstract: Information about heart failure with reduced ejection fraction (HFrEF) in women and the potential effects of aging in the female heart is scarce. We investigated the vulnerability to develop HFrEF in female elderly mice compared to young animals, as well as potential differences in reverse remodeling. First, HF was induced by isoproterenol infusion (30 mg/kg/day, 28 days) in young (10-week-old) and elderly (22-month-old) female mice. In a second set of animals, mice underwent isoproterenol infusion followed by no treatment during 28 additional days. Cardiac remodeling was assessed by echocardiography, histology and gene expression of collagen-I and collagen-III. Following isoproterenol infusion, elderly mice developed similar HFrEF features compared to young animals, except for greater cell hypertrophy and tissue fibrosis. After beta-adrenergic withdrawal, young female mice experienced complete reversal of the HFrEF phenotype. Conversely, reversed remodeling was impaired in elderly animals, with no significant recovery of LV ejection fraction, cardiomyocyte hypertrophy and collagen deposition. In conclusion, chronic isoproterenol infusion is a valid HF model for elderly and young female mice and induces a similar HF phenotype in both. Elderly animals, unlike young, show impaired reverse remodeling, with persistent tissue fibrosis and cardiac dysfunction even after beta-adrenergic withdrawal

Lung Extracellular Matrix Hydrogels-Derived Vesicles Contribute to Epithelial Lung Repair

The use of physiomimetic decellularized extracellular matrix-derived hydrogels is attracting interest since they can modulate the therapeutic capacity of numerous cell types, including mesenchymal stromal cells (MSCs). Remarkably, extracellular vesicles (EVs) derived from MSCs display similar functions as their parental cells, mitigating tissue damage in lung diseases. However, recent data have shown that ECM-derived hydrogels could release other resident vesicles similar to EVs. Here, we aim to better understand the contribution of EVs and ECM-vesicles released from MSCs and/or lung-derived hydrogel (L-HG) in lung repair by using an in vitro lung injury model. L-HG derived-vesicles and MSCs EVs cultured either in L-HG or conventional plates were isolated and characterized. The therapeutic capacity of vesicles obtained from each experimental condition was tested by using an alveolar epithelial wound-healing assay. The number of ECM-vesicles released from acellular L-HG was 10-fold greater than EVs from conventional MSCs cell culture revealing that L-HG is an important source of bioactive vesicles. MSCs-derived EVs and L-HG vesicles have similar therapeutic capacity in lung repair. However, when wound closure rate was normalized by total proteins, the MSCs-derived EVs shows higher therapeutic potential to those released by L-HG. The EVs released from L-HG must be considered when HG is used as substrate for cell culture and EVs isolation

A Fast and Efficient Decellularization Method for Tissue Slices

The study and use of decellularized extracellular matrix (dECM) in tissue engineering, regenerative medicine, and pathophysiology have become more prevalent in recent years. To obtain dECM, numerous decellularization procedures have been developed for the entire organ or tissue blocks, employing either perfusion of decellularizing agents through the tissue's vessels or submersion of large sections in decellularizing solutions. However, none of these protocols are suitable for thin tissue slices (less than 100 μm) or allow side-by-side analysis of native and dECM consecutive tissue slices. Here, we present a detailed protocol to decellularize tissue sections while maintaining the sample attached to a glass slide. This protocol consists of consecutive washes and incubations of simple decellularizing agents: ultrapure water, sodium deoxycholate (SD) 2%, and deoxyribonuclease I solution 0.3 mg/mL (DNase I). This novel method has been optimized for a faster decellularization time (2-3 h) and a better correlation between dECM properties and native tissue-specific biomarkers, and has been tested in different types of tissues and species, obtaining similar results. Furthermore, this method can be used for scarce and valuable samples such as clinical biopsies

Hypothalamic Regulation of Liver and Muscle Nutrient Partitioning by Brain-Specific Carnitine Palmitoyltransferase 1C in Male Mice

Carnitine palmitoyltransferase (CPT) 1C, a brain-specific protein localized in the endoplasmic reticulum of neurons, is expressed in almost all brain regions. Based on global knockout (KO) models, CPT1C has demonstrated relevance in hippocampus-dependent spatial learning and in hypothalamic regulation of energy balance. Specifically, it has been shown that CPT1C is protective against high-fat diet-induced obesity (DIO), and that CPT1C KO mice show reduced peripheral fatty acid oxidation (FAO) during both fasting and DIO. However, the mechanisms mediating CPT1C-dependent regulation of energy homeostasis remain unclear. Here, we focus on the mechanistic understanding of hypothalamic CPT1C on the regulation of fuel selection in liver and muscle of male mice during energy deprivation situations, such as fasting. In CPT1C-deficient mice, modulation of the main hypothalamic energy sensors (50 adenosine monophosphate-activated protein kinase, Sirtuin 1, and mammalian target of rapamycin) was impaired and plasma catecholamine levels were decreased. Consequently, CPT1C-deficient mice presented defective fasting-induced FAO in liver, leading to higher triacylglycerol accumulation and lower glycogen levels. Moreover, muscle pyruvate dehydrogenase activity was increased, which was indicative of glycolysis enhancement. The respiratory quotient did not decrease in CPT1CKO mice after 48 hours of fasting, confirming a defective switch on fuel substrate selection under hypoglycemia. Phenotype reversion studies identified the mediobasal hypothalamus (MBH) as the main area mediating CPT1C effects on fuel selection. Overall, our data demonstrate that CPT1C in the MBH is necessary for proper hypothalamic sensing of a negative energy balance and fuel partitioning in liver and muscle

Development of a physiomimetic model of acute respiratory distress syndrome by using ECM hydrogels and organ-on-a-chip devices

Acute Respiratory Distress Syndrome is one of the more common fatal complications in COVID-19, characterized by a highly aberrant inflammatory response. Pre-clinical models to study the effect of cell therapy and anti-inflammatory treatments have not comprehensively reproduced the disease due to its high complexity. This work presents a novel physiomimetic in vitro model for Acute Respiratory Distress Syndrome using lung extracellular matrix-derived hydrogels and organ-on-a-chip devices. Monolayres of primary alveolar epithelial cells were cultured on top of decellullarized lung hydrogels containing primary lung mesenchymal stromal cells. Then, cyclic stretch was applied to mimic breathing, and an inflammatory response was induced by using a bacteriotoxin hit. Having simulated the inflamed breathing lung environment, we assessed the effect of an anti-inflammatory drug (i.e., dexamethasone) by studying the secretion of the most relevant inflammatory cytokines. To better identify key players in our model, the impact of the individual factors (cyclic stretch, decellularized lung hydrogel scaffold, and the presence of mesenchymal stromal cells) was studied separately. Results showed that developed model presented a more reduced inflammatory response than traditional models, which is in line with what is expected from the response commonly observed in patients. Further, from the individual analysis of the different stimuli, it was observed that the use of extracellular matrix hydrogels obtained from decellularized lungs had the most significant impact on the change of the inflammatory response. The developed model then opens the door for further in vitro studies with a better-adjusted response to the inflammatory hit and more robust results in the test of different drugs or cell therapy

Hypothalamic ceramide levels regulated by CPT1C mediate the orexigenic effect of ghrelin.

Recent data suggest that ghrelin exerts its orexigenic action through regulation of hypothalamic AMP-activated protein kinase (AMPK) pathway, leading to a decline in malonyl-CoA levels and desinhibition of carnitine palmitoyltransferase 1A (CPT1A), which increases mitochondrial fatty acid oxidation and ultimately enhances the expression of the orexigenic neuropeptides agouti-related protein (AgRP) and neuropeptide Y (NPY). However, it is unclear whether the brain-specific isoform CPT1C, which is located in the endoplasmic reticulum (ER) of neurons, may play a role on this action. Here, we demonstrate that ghrelin's orexigenic action is totally blunted in CPT1C knock-out (KO) mice, despite having the canonical ghrelin signaling pathway activated. We also demonstrate that ghrelin elicits a marked upregulation of hypothalamic C18:0-ceramide levels mediated by CPT1C. Notably, central inhibition of ceramide synthesis with myriocin negated ghrelin's orexigenic action and normalized the levels of AgRP and NPY, as well as their key transcription factors pCREB and FoxO1. Finally, central treatment with ceramide induced food intake and orexigenic neuropeptides expression in CPT1C KO mice. Overall, these data indicate that, in addition to formerly reported mechanisms, ghrelin also induces food intake through regulation of hypothalamic CPT1C and ceramide metabolism, a finding of potential importance for the understanding and treatment of obesity

CPT1C in the ventromedial nucleus of the hypothalamus is necessary for brown fat thermogenesis activation in obesity

OBJECTIVE: Carnitine palmitoyltransferase 1C (CPT1C) is implicated in central regulation of energy homeostasis. Our aim was to investigate whether CPT1C in the ventromedial nucleus of the hypothalamus (VMH) is involved in the activation of brown adipose tissue (BAT) thermogenesis in the early stages of diet-induced obesity. METHODS: CPT1C KO and wild type (WT) mice were exposed to short-term high-fat (HF) diet feeding or to intracerebroventricular leptin administration and BAT thermogenesis activation was evaluated. Body weight, adiposity, food intake, and leptinemia were also assayed. RESULTS: Under 7 days of HF diet, WT mice showed a maximum activation peak of BAT thermogenesis that counteracted obesity development, whereas this activation was impaired in CPT1C KO mice. KO animals evidenced higher body weight, adiposity, hyperleptinemia, ER stress, and disrupted hypothalamic leptin signaling. Leptin-induced BAT thermogenesis was abolished in KO mice. These results indicate an earlier onset leptin resistance in CPT1C KO mice. Since AMPK in the VMH is crucial in the regulation of BAT thermogenesis, we analyzed if CPT1C was a downstream factor of this pathway. Genetic inactivation of AMPK within the VMH was unable to induce BAT thermogenesis and body weight loss in KO mice, indicating that CPT1C is likely downstream AMPK in the central mechanism modulating thermogenesis within the VMH. Quite opposite, the expression of CPT1C in the VMH restored the phenotype. CONCLUSION: CPT1C is necessary for the activation of BAT thermogenesis driven by leptin, HF diet exposure, and AMPK inhibition within the VMH. This study underscores the importance of CPT1C in the activation of BAT thermogenesis to counteract diet-induced obesity. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved. KEYWORDS: Brown adipose tissue; CPT1C; Diet-induced obesity; Hypothalamus; Thermogenesi

Ghrelin causes a decline in GABA release by reducing fatty acid oxidation in cortex

Lipid metabolism, specifically fatty acid oxidation (FAO) mediated by carnitine palmitoyltransferase (CPT) 1A, has been described to be an important actor of ghrelin action in hypothalamus. However, it is not known whether CPT1A and FAO mediate the effect of ghrelin on the cortex. Here, we show that ghrelin produces a differential effect on CPT1 activity and γ-aminobutyric acid (GABA) metabolism in the hypothalamus and cortex of mice. In the hypothalamus, ghrelin enhances CPT1A activity while GABA transaminase (GABAT) activity, a key enzyme in GABA shunt metabolism, is unaltered. However, in cortex CPT1A activity and GABAT activity are reduced after ghrelin treatment. Furthermore, in primary cortical neurons, ghrelin reduces GABA release through a CPT1A reduction. By using CPT1A floxed mice, we have observed that genetic ablation of CPT1A recapitulates the effect of ghrelin on GABA release in cortical neurons, inducing reductions in mitochondrial oxygen consumption, cell content of citrate and α-ketoglutarate, and GABA shunt enzyme activity. Taken together, these observations indicate that ghrelin-induced changes in CPT1A activity modulate mitochondrial function, yielding changes in GABA metabolism. This evidence suggests that the action of ghrelin on GABA release is region specific within the brain, providing a basis for differential effects of ghrelin in the central nervous system. Keywords: Ghrelin, GABA, Fatty acid oxidation, CPT1A, Cortical neuron

A cluster-randomized trial of hydroxychloroquine for prevention of Covid-19

Background: current strategies for preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are limited to nonpharmacologic interventions. Hydroxychloroquine has been proposed as a postexposure therapy to prevent coronavirus disease 2019 (Covid-19), but definitive evidence is lacking. Methods: we conducted an open-label, cluster-randomized trial involving asymptomatic contacts of patients with polymerase-chain-reaction (PCR)-confirmed Covid-19 in Catalonia, Spain. We randomly assigned clusters of contacts to the hydroxychloroquine group (which received the drug at a dose of 800 mg once, followed by 400 mg daily for 6 days) or to the usual-care group (which received no specific therapy). The primary outcome was PCR-confirmed, symptomatic Covid-19 within 14 days. The secondary outcome was SARS-CoV-2 infection, defined by symptoms compatible with Covid-19 or a positive PCR test regardless of symptoms. Adverse events were assessed for up to 28 days. Results: the analysis included 2314 healthy contacts of 672 index case patients with Covid-19 who were identified between March 17 and April 28, 2020. A total of 1116 contacts were randomly assigned to receive hydroxychloroquine and 1198 to receive usual care. Results were similar in the hydroxychloroquine and usual-care groups with respect to the incidence of PCR-confirmed, symptomatic Covid-19 (5.7% and 6.2%, respectively; risk ratio, 0.86 [95% confidence interval, 0.52 to 1.42]). In addition, hydroxychloroquine was not associated with a lower incidence of SARS-CoV-2 transmission than usual care (18.7% and 17.8%, respectively). The incidence of adverse events was higher in the hydroxychloroquine group than in the usual-care group (56.1% vs. 5.9%), but no treatment-related serious adverse events were reported. Conclusions: postexposure therapy with hydroxychloroquine did not prevent SARS-CoV-2 infection or symptomatic Covid-19 in healthy persons exposed to a PCR-positive case patient. (Funded by the crowdfunding campaign YoMeCorono and others; BCN-PEP-CoV2 ClinicalTrials.gov number, NCT04304053.)

Analysis of monitoring programmes and their suitability for ecotoxicological risk assessment in four Spanish basins

Data from four Spanish basin management authorities were analysed. Chemical and biological data from four Spanish basin management authorities were analysed, focusing on three consecutive years. Aims were to i) determine the chemicals most likely responsible for the environmental toxicological risk in the four Spanish basins and ii) investigate the relationships between toxicological risk and biological status in these catchments. The toxicological risk of chemicals was evaluated using the toxic unit (TU) concept. With these data we considered if the potential risk properly reflects the risk to the community or, alternatively, if new criteria should be developed to improve risk assessment. Data study revealed inadequacies in processing and monitoring that should be improved (e.g., site coincidence for chemical and biological sampling). Analysis of the chemical data revealed high potential toxicological risk in the majority of sampling points, to which metals were the main contributors to this risk. However, clear relationships between biological quality and chemical risk were found only in one river. Further investigation of metal toxicity may be necessary, and future analyses are necessary to accurately estimate the risk to the environment.The present work was funded by the Spanish Ministry of Economics and Competitiveness through the Consolider-Ingenio 2010 Program (project Scarce CSD2009-00065). The authors would like to acknowledge the Confederacion Hidrografica del Ebro (C. Duran and V. Sanchez-Tello), Confederacion Hidrografica del Guadalquivir (V. Cifuentes), Agenda Andaluza del Agua, Agenda Catalana de l'Aigua (A Munne, L Tirapu) and Confederacion Hidrografica del Jucar (MA Pinon) which kindly provided the monitoring data. Nuria De Castro-Catala holds a predoctoral grant from the University of Barcelona. We would like to thank two anonymous reviewers for their suggestions which have improved this manuscript.López Doval, JC.; De Castro Catala, N.; Andrés Doménech, I.; Blasco, J.; Ginebreda, A.; Muñoz, I. (2012). Analysis of monitoring programmes and their suitability for ecotoxicological risk assessment in four Spanish basins. Science of the Total Environment. 440:194-203. https://doi.org/10.1016/j.scitotenv.2012.07.035S19420344