T-1-MRI Fluorescent Iron Oxide Nanoparticles by Microwave Assisted Synthesis

Iron oxide nanoparticles have long been studied as a T-2 contrast agent in MRI due to their superparamagnetic behavior. T-1-based positive contrast, being much more favorable for clinical application due to brighter and more accurate signaling is, however, still limited to gadolinium- or manganese-based imaging tools. Though being the only available commercial positive-contrast agents, they lack an efficient argument when it comes to biological toxicity and their circulatory half-life in blood. The need arises to design a biocompatible contrast agent with a scope for easy surface functionalization for long circulation in blood and/or targeted imaging. We hereby propose an extremely fast microwave synthesis for fluorescein-labeled extremely-small iron oxide nanoparticles (fdIONP), in a single step, as a viable tool for cell labeling and T-1-MRI. We demonstrate the capabilities of such an approach through high-quality magnetic resonance angiographic images of mice.This study was supported by a grant from Ministerio de Economia y Competitividad (MAT2013-47303-P) and European Union (Cardionext, ITN-FP7-608027).S

Superparamagnetic Nanoparticles for Atherosclerosis Imaging

The production of magnetic nanoparticles of utmost quality for biomedical imaging requires several steps, from the synthesis of highly crystalline magnetic cores to the attachment of the different molecules on the surface. This last step probably plays the key role in the production of clinically useful nanomaterials. The attachment of the different biomolecules should be performed in a defined and controlled fashion, avoiding the random adsorption of the components that could lead to undesirable byproducts and ill-characterized surface composition. In this work, we review the process of creating new magnetic nanomaterials for imaging, particularly for the detection of atherosclerotic plaque, in vivo. Our focus will be in the different biofunctionalization techniques that we and several other groups have recently developed. Magnetic nanomaterial functionalization should be performed by chemoselective techniques. This approach will facilitate the application of these nanomaterials in the clinic, not as an exception, but as any other pharmacological compound.The authors thank the Spanish Ministry of Science (SAF2011-25445), the Comunidad de Madrid (S2010/BMD-2326, Inmunothercan-CM, NANOCOPD-CIBERES-CIBERBBN-SEPAR), and the EU 7th Framework Program (FP7-PEOPLE-ITN-264864 Pinet and FP7-PEOPLE-2013-ITN CardioNext).S

Phosphatidylcholine-coated iron oxide nanomicelles for in vivo prolonged circulation time with an antibiofouling protein corona

We report the synthesis of micellar phosphatidylcholine-coated superparamagnetic iron oxide nanoparticles as a new long circulation contrast agents for magnetic resonance imaging. Oleic acid-coated Fe3 O4 nanoparticles were first prepared through thermal degradation and then encapsulated into small clusters with a phosphatidylcholine coating to obtain hydrophilic nanomicelles. A thorough characterization confirmed the chemical nature of the coating and the excellent colloidal stability of these nanomicelles in aqueous media. Magnetization and relaxivity properties proved their suitability as magnetic resonance imaging (MRI) contrast agent and in vitro cell viability data showed low toxicity. Vascular lifetime and elimination kinetics in the liver were assessed by blood relaxometry and by in vivo MRI in rats and compared with "control" particles prepared with a polyethylene glycol derivative. These micellar particles had a lifetime in blood of more than 10 h, much longer than the control nanoparticles (≈2 h), which is remarkable considering that the coating molecule is a small biocompatible zwitterionic phospholipid. The protein corona was characterized after incubation with rat serum at different times by high-throughput proteomics, showing a higher proportion of bound apolipoproteins and other dysopsonins for the phosphatidylcholine particles. The antibiofouling properties of this corona and its resistance to the adsorption of proteins corroborate the observed enhanced stability and prolonged systemic circulation.This study is supported by a grant from FP7 Marie Curie, Pulmonary imaging network (PINET), by Fundacio La Maratode TV3 (70/C/2012) and by a grant from the Comunidad de Madrid (S2010/BMD-2326, Inmunothercan-CM) and by Spanish Economy Ministry (MAT2013-47303-P). We thank E. Urones (Centro Nacional de Microscopia de la Universidad Complutense de Madrid) for the transmission electronic microscopy imaging; P. Morales (Instituto de Ciencia de Materiales de la Universidad Autonoma de Madrid) for the thermogravimetric and magnetization analysis and B. Salinas (Fundacion Centro Nacional de Investigaciones Cardiovasculares and CIBER de Enfermedades Respiratorias) for the TEM picture of oleic acid coated Fe3 O4. The authors declare no competing financial interests.S

Comprehensive Quantification of the Modified Proteome Reveals Oxidative Heart Damage in Mitochondrial Heteroplasmy

Post-translational modifications hugely increase the functional diversity of proteomes. Recent algorithms based on ultratolerant database searching are forging a path to unbiased analysis of peptide modifications by shotgun mass spectrometry. However, these approaches identify only one-half of the modified forms potentially detectable and do not map the modified residue. Moreover, tools for the quantitative analysis of peptide modifications are currently lacking. Here, we present a suite of algorithms that allows comprehensive identification of detectable modifications, pinpoints the modified residues, and enables their quantitative analysis through an integrated statistical model. These developments were used to characterize the impact of mitochondrial heteroplasmy on the proteome and on the modified peptidome in several tissues from 12-week-old mice. Our results reveal that heteroplasmy mainly affects cardiac tissue, inducing oxidative damage to proteins of the oxidative phosphorylation system, and provide a molecular mechanism explaining the structural and functional alterations produced in heart mitochondria.We thank Simon Bartlett (CNIC) for English editing. This study was supported by competitive grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (BIO2015-67580-P) through the Carlos III Institute of Health-Fondo de Investigacion Sanitaria (PRB2, IPT13/0001-ISCIII-SGEFI/FEDER; ProteoRed), by Fundacion La Marato TV3, and by FP7-PEOPLE-2013-ITN ``Next-Generation Training in Cardiovascular Research and Innovation-Cardionext.'' N.B. is a FP7-PEOPLE-2013-ITN-Cardionext Fellow. The CNIC is supported by the MINECO and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO Award SEV-2015-0505).S

In vivo imaging of lung inflammation with neutrophil-specific Ga-68 nano-radiotracer

In vivo detection and quantification of inflammation is a major goal in molecular imaging. Furthermore, cell-specific detection of inflammation would be a tremendous advantage in the characterization of many diseases. Here, we show how this goal can be achieved through the synergistic combination of nanotechnology and nuclear imaging. One of the most remarkable features of this hybrid approach is the possibility to tailor the pharmacokinetics of the nanomaterial-incorporated biomolecule and radionuclide. A good example of this approach is the covalent binding of a large amount of a neutrophil-specific, hydrophobic peptide on the surface of Ga-68 core-doped nanoparticles. This new nano-radiotracer has been used for non-invasive in vivo detection of acute inflammation with very high in vivo labelling efficiency, i.e. a large percentage of labelled neutrophils. Furthermore, we demonstrate that the tracer is neutrophil-specific and yields images of neutrophil recruitment of unprecedented quality. Finally, the nano-radiotracer was successfully detected in chronic inflammation in atherosclerosis-prone ApoE(-/-) mice after several weeks on a high-fat diet

NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice

While NLRP3‐inflammasome has been implicated in cardiovascular diseases, its role in physiological cardiac aging is largely unknown. During aging, many alterations occur in the organism, which are associated with progressive impairment of metabolic pathways related to insulin resistance, autophagy dysfunction, and inflammation. Here, we investigated the molecular mechanisms through which NLRP3 inhibition may attenuate cardiac aging. Ablation of NLRP3‐inflammasome protected mice from age‐related increased insulin sensitivity, reduced IGF‐1 and leptin/adiponectin ratio levels, and reduced cardiac damage with protection of the prolongation of the agedependent PR interval, which is associated with atrial fibrillation by cardiovascular aging and reduced telomere shortening. Furthermore, old NLRP3 KO mice showed an inhibition of the PI3K/AKT/mTOR pathway and autophagy improvement, compared with old wild mice and preserved Nampt‐mediated NAD+ levels with increased SIRT1 protein expression. These findings suggest that suppression of NLRP3 prevented many age‐associated changes in the heart, preserved cardiac function of aged mice and increased lifespan.Andalusian regional government; Consejería de Salud de la Junta de Andalucia, Grant/ Award Number: PI‐0036‐2014; Ministerio de economía y competitividad, Grant/Award Number: SAF2017‐84494‐C2‐1‐

A Neutrophil Timer Coordinates Immune Defense and Vascular Protection

Neutrophils eliminate pathogens efficiently but can inflict severe damage to the host if they over-activate within blood vessels. It is unclear how immunity solves the dilemma of mounting an efficient anti-microbial defense while preserving vascular health. Here, we identify a neutrophil-intrinsic program that enabled both. The gene Bmal1 regulated expression of the chemokine CXCL2 to induce chemokine receptor CXCR2-dependent diurnal changes in the transcriptional and migratory properties of circulating neutrophils. These diurnal alterations, referred to as neutrophil aging, were antagonized by CXCR4 (C-X-C chemokine receptor type 4) and regulated the outer topology of neutrophils to favor homeostatic egress from blood vessels at night, resulting in boosted anti-microbial activity in tissues. Mice engineered for constitutive neutrophil aging became resistant to infection, but the persistence of intravascular aged neutrophils predisposed them to thrombo-inflammation and death. Thus, diurnal compartmentalization of neutrophils, driven by an internal timer, coordinates immune defense and vascular protection. Neutrophils display circadian oscillations in numbers and phenotype in the circulation. Adrover and colleagues now identify the molecular regulators of neutrophil aging and show that genetic disruption of this process has major consequences in immune cell trafficking, anti-microbial defense, and vascular health.This study was supported by Intramural grants from A∗STAR to L.G.N., BES-2013-065550 to J.M.A., BES-2010-032828 to M.C.-A, and JCI-2012-14147 to L.A.W (all from Ministerio de Economía, Industria y Competitividad; MEIC). Additional MEIC grants were SAF2014-61993-EXP to C.L.-R.; SAF2015-68632-R to M.A.M. and SAF-2013-42920R and SAF2016-79040Rto D.S. D.S. also received 635122-PROCROP H2020 from the European Commission and ERC CoG 725091 from the European Research Council (ERC). ERC AdG 692511 PROVASC from the ERC and SFB1123-A1 from the Deutsche Forschungsgemeinschaft were given to C.W.; MHA VD1.2/81Z1600212 from the German Center for Cardiovascular Research (DZHK) was given to C.W. and O.S.; SFB1123-A6 was given to O.S.; SFB914-B08 was given to O.S. and C.W.; and INST 211/604-2, ZA 428/12-1, and ZA 428/13-1 were given to A.Z. This study was also supported by PI12/00494 from Fondo de Investigaciones Sanitarias (FIS) to C.M.; PI13/01979, Cardiovascular Network grant RD 12/0042/0054, and CIBERCV to B.I.; SAF2015-65607-R, SAF2013-49662-EXP, and PCIN-2014-103 from MEIC; and co-funding by Fondo Europeo de Desarrollo Regional (FEDER) to A.H. The CNIC is supported by the MEIC and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505)

A Neutrophil Timer Coordinates Immune Defense and Vascular Protection

Neutrophils eliminate pathogens efficiently but can inflict severe damage to the host if they over-activate within blood vessels. It is unclear how immunity solves the dilemma of mounting an efficient anti-microbial defense while preserving vascular health. Here, we identify a neutrophil-intrinsic program that enabled both. The gene Bmal1 regulated expression of the chemokine CXCL2 to induce chemokine receptor CXCR2-dependent diurnal changes in the transcriptional and migratory properties of circulating neutrophils. These diurnal alterations, referred to as neutrophil aging, were antagonized by CXCR4 (C-X-C chemokine receptor type 4) and regulated the outer topology of neutrophils to favor homeostatic egress from blood vessels at night, resulting in boosted anti-microbial activity in tissues. Mice engineered for constitutive neutrophil aging became resistant to infection, but the persistence of intravascular aged neutrophils predisposed them to thrombo-inflammation and death. Thus, diurnal compartmentalization of neutrophils, driven by an internal timer, coordinates immune defense and vascular protection.We thank all members of the Hidalgo Lab for discussion and insightful comments; J.M. Ligos, R. Nieto, and M. Viton for help with sorting and cytometric analyses; I. Ortega and E. Santos for animal husbandry; D. Rico, M.J. Gomez, C. Torroja, and F. Sanchez-Cabo for insightful comments and help with transcriptomic analyses; V. Labrador, E. Arza, A.M. Santos, and the Microscopy Unit of the CNIC for help with microscopy; S. Aznar-Benitah, U. Albrecht, Q.-J. Meng, B. Staels, and H. Duez for the generous gift of mice; J.A. Enriquez and J. Avila for scientific insights; and J.M. Garcia and A. Diez de la Cortina for art. This study was supported by Intramural grants from A* STAR to L.G.N., BES-2013-065550 to J.M.A., BES-2010-032828 to M.C.-A, and JCI-2012-14147 to L.A.W (all from Ministerio de Economia, Industria y Competitividad; MEIC). Additional MEIC grants were SAF2014-61993-EXP to C.L.-R.; SAF2015-68632-R to M.A.M. and SAF-2013-42920R and SAF2016-79040Rto D.S. D.S. also received 635122-PROCROP H2020 from the European Commission and ERC CoG 725091 from the European Research Council (ERC). ERC AdG 692511 PROVASC from the ERC and SFB1123-A1 from the Deutsche Forschungsgemeinschaft were given to C.W.; MHA VD1.2/81Z1600212 from the German Center for Cardiovascular Research (DZHK) was given to C.W. and O.S.; SFB1123-A6 was given to O.S.; SFB914-B08 was given to O.S. and C.W.; and INST 211/604-2, ZA 428/12-1, and ZA 428/13-1 were given to A.Z. This study was also supported by PI12/00494 from Fondo de Investigaciones Sanitarias (FIS) to C.M.; PI13/01979, Cardiovascular Network grant RD 12/0042/0054, and CIBERCV to B.I.; SAF2015-65607-R, SAF2013-49662-EXP, and PCIN-2014-103 from MEIC; and co-funding by Fondo Europeo de Desarrollo Regional (FEDER) to A.H. The CNIC is supported by the MEIC and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (MEIC award SEV-2015-0505).S

Mitochondrial OxPhos: the integrator of the cellular metabolic status

The oxidative phosphorylation (OXPHOS) system is the only process in animal cells with components encoded by two genomes, mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). MtDNA is polyploid, maternally inherited, suffers marginal recombination and has a rate of mutation which is one order of magnitude higher than nDNA, thus resulting in high variability of healthy mtDNA haplotypes. Nuclear OXPHOS genes present lower mutation rate and recombination. However they have alternative options due to allele variants and tissue-specific variants, which confront identical nDNA with diverse mtDNA within the same individual. This asymmetry leads to the physical match constraint: the fact that mtDNA-encoded proteins have to physically assemble with the nuclearencoded ones to build the respiratory complexes. All mtDNAs of a given cell are essentially identical, a situation termed homoplasmy. Heteroplasmy refers to the presence of more than one variant of mtDNA co-existing in the same cytoplasm. Two major interconnected questions remain to be solved: (i) are different versions of wild-type mtDNAs interchangeable without any phenotypic impact? And, (ii) does the iatrogenic introduction of heteroplasmy result in health risks for the offspring by triggering a genomic conflict between alternative mtDNA variants with potential negative consequences? We intend to understand the long-term consequences of mtDNA/nDNA mismatch and heteroplasmy. In particular, (i) whether or not modifications in key nuclear genes involved in mitochondrial function modulate the impact of alternative mtDNAs, (ii) whether or not the co-existence of two wild-type mtDNAs in the same cytoplasm impacts on metabolism, and (iii) the identification of the signals involved in regulation. To address these questions, we use conplastic mice (identical nucleus but interchanged mtDNAs, C57 and NZB non-pathological variants) and heteroplasmic mice (co-existence of both mtDNAs in the same cell). Here, we systematically characterised conplastic mice throughout embryo development and during their lifespan through omics techniques and physiological and phenotyping studies. We report that the mtDNA haplotype profoundly influences the reactive oxygen species generation, energy homeostasis, metabolism and ageing parameters among others, resulting in different healthy longevities of conplastic strains. Our recent analysis supports: (1) mtDNA variants induce functional OXPHOS differences under the same nuclear background. (2) Cells can adapt their OXPHOS performance to generate healthy animals, regardless the nDNA/mtDNA match. (3) Different cell types show specific sensitivity to the nDNA/mtDNA match. (4) Changes in the nDNA/mtDNA match induce significant metabolic differences that are manifested in the adulthood and which dramatically impact on ageing and longevity. Heteroplasmy is actively combated by several mechanisms, including the degradation of the paternal mtDNA upon fertilization, and the existence of a genetic mtDNA bottleneck in oocyte development. Heteroplasmy can be naturally generated by mutagenesis during mtDNA replication, but also can originate from novel medical technologies aiming (i) to prevent the transmission of mtDNA-linked diseases, (ii) to improve the fertility by human oocyte “rejuvenation”, and (iii) to restore the function of damaged cells using the transfer of foreign mitochondria. Here we show that mtDNA heteroplasmy modulates the viability and metabolism of embryonic cells, as well as their ability to become iPS. During post-natal life, most of the tissues can remove the remaining heteroplasmy and preserve life-long fitness. In contrast, critical tissues like cardiac and skeletal muscles as well as the lung maintain heteroplasmy and suffer a progressive metabolic stress leading to a severe adult pathologies, including cardiopulmonary failure, skeletal muscle wasting, frailty and premature death.S

T1-MRI Fluorescent Iron Oxide Nanoparticles by Microwave Assisted Synthesis

Iron oxide nanoparticles have long been studied as a T2 contrast agent in MRI due to their superparamagnetic behavior. T1-based positive contrast, being much more favorable for clinical application due to brighter and more accurate signaling is, however, still limited to gadolinium- or manganese-based imaging tools. Though being the only available commercial positive-contrast agents, they lack an efficient argument when it comes to biological toxicity and their circulatory half-life in blood. The need arises to design a biocompatible contrast agent with a scope for easy surface functionalization for long circulation in blood and/or targeted imaging. We hereby propose an extremely fast microwave synthesis for fluorescein-labeled extremely-small iron oxide nanoparticles (fdIONP), in a single step, as a viable tool for cell labeling and T1-MRI. We demonstrate the capabilities of such an approach through high-quality magnetic resonance angiographic images of mice