Microwave-driven synthesis of bisphosphonate nanoparticles allows in vivo visualisation of atherosclerotic plaque

A fast and reproducible microwave-driven process has allowed us to synthesise neridronate-functionalised nanoparticles. Contrary to tradition, the phosphate groups decorate the outside layer of the particles providing Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. In vivo and ex vivo detection by T2-weighted MRI is demonstrated and validated by histology. The accumulation in the plaque takes place in less than one hour following the intravenous injection, which is particularly suitable for clinical applications

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

Cell identity and nucleo-mitochondrial genetic context modulate OXPHOS performance and determine somatic heteroplasmy dynamics

Heteroplasmy, multiple variants of mitochondrial DNA (mtDNA) in the same cytoplasm, may be naturally generated by mutations but is counteracted by a genetic mtDNA bottleneck during oocyte development. Engineered heteroplasmic mice with nonpathological mtDNA variants reveal a nonrandom tissue-specific mtDNA segregation pattern, with few tissues that do not show segregation. The driving force for this dynamic complex pattern has remained unexplained for decades, challenging our understanding of this fundamental biological problem and hindering clinical planning for inherited diseases. Here, we demonstrate that the nonrandom mtDNA segregation is an intracellular process based on organelle selection. This cell type-specific decision arises jointly from the impact of mtDNA haplotypes on the oxidative phosphorylation (OXPHOS) system and the cell metabolic requirements and is strongly sensitive to the nuclear context and to environmental cues

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

One-step fast synthesis of nanoparticles for MRI: coating chemistry as the key variable determining positive or negative contrast

Iron oxide nanomaterial is a typical example of a magnetic resonance imaging probe for negative contrast. It has also been shown how this nanomaterial can be synthesized for positive contrast by modification of the composition and size of the core. However, the role of the organic coating in the relaxometric properties is largely unexplored. Here, maghemite nanoparticles with either excellent positive or very good negative contrast performance are obtained by modifying coating thickness while the core is kept unchanged. Different nanoparticles with tailored features as contrast agent according to the coating layer thickness have been obtained in a single-step microwave-driven synthesis by heating at different temperatures. A comprehensive analysis is conducted of how the composition and structure of the coating affects the final magnetic, relaxometric, and imaging performance. These results show how the organic coating plays a fundamental role in the intrinsic relaxometric parameters of iron oxide-based contrast media.This study was supported by a grant from the Spanish Ministry for Economy and Competitiveness (MEyC) (grant nos. MAT2013-47303-P, MAT2014-52069-R, SAF2016-79593-P). L.G. received financial support from the Ramón y Cajal subprogram (RYC-2014-15512). The CNIC is supported by the MEyC and the Pro-CNIC Foundation and is a SO-Center of Excellence (SEV-2015-0505)

One-Step Fast Synthesis of Nanoparticles for MRI: Coating Chemistry as the Key Variable Determining Positive or Negative Contrast

Iron oxide nanomaterial is a typical example of a magnetic resonance imaging probe for negative contrast. It has also been shown how this nanomaterial can be synthesized for positive contrast by modification of the composition and size of the core. However, the role of the organic coating in the relaxometric properties is largely unexplored. Here, maghemite nanoparticles with either excellent positive or very good negative contrast performance are obtained by modifying coating thickness while the core is kept unchanged. Different nanoparticles with tailored features as contrast agent according to the coating layer thickness have been obtained in a single-step microwave-driven synthesis by heating at different temperatures. A comprehensive analysis is conducted of how the composition and structure of the coating affects the final magnetic, relaxometric, and imaging performance. These results show how the organic coating plays a fundamental role in the intrinsic relaxometric parameters of iron oxide-based contrast media

Screening of effective pharmacological treatments for MELAS syndrome using yeasts, fibroblasts and cybrid models of the disease

Trabajo presentado como póster al 22nd IUBMB & 37th FEBS Congress, celebrado en Sevilla (España) del 4 al 9 de septiembre de 2012.[Backgroun and Purpose]: MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease most usually caused by point mutations in tRNA genes encoded by mitochondrial DNA (mtDNA). Approximately 80% of cases of MELAS syndrome are associated with a m.3243A > G mutation in the MT-TL1 gene, which encodes the mitochondrial tRNALeu (UUR). Currently, no effective treatments are available for this chronic progressive disorder. Treatment strategies in MELAS and other mitochondrial diseases consist of several drugs that diminish the deleterious effects of the abnormal respiratory chain function, reduce the presence of toxic agents or correct deficiencies in essential cofactors.[Experimental Approach]: We evaluated the effectiveness of some common pharmacological agents that have been utilized in the treatment of MELAS, in yeast, fibroblast and cybrid models of the disease. The yeast model harbouring the A14G mutation in the mitochondrial tRNALeu(UUR) gene, which is equivalent to the A3243G mutation in humans, was used in the initial screening. Next, the most effective drugs that were able to rescue the respiratory deficiency in MELAS yeast mutants were tested in fibroblasts and cybrid models of MELAS disease.[Key Results]: According to our results, supplementation with riboflavin or coenzyme Q10 effectively reversed the respiratory defect in MELAS yeast and improved the pathologic alterations in MELAS fibroblast and cybrid cell models.[Conclusions and Implications]: Our results indicate that cell models have great potential for screening and validating the effects of novel drug candidates for MELAS treatment and presumably also for other diseases with mitochondrial impairment.This work was supported by FIS PI10/00543 grant, FIS EC08/00076 grant, Ministerio de Sanidad, Spain and Fondo Europeo de Desarrollo Regional (FEDER-Unión Europea), SAS111242 grant, Servicio Andaluz de Salud-Junta de Andalucía, Proyecto de Investigación de Excelencia de la Junta de Andalucía CTS-5725, and by AEPMI (Asociación de Enfermos de Patología Mitocondrial), FEEL (Fundación Española de Enfermedades Lisosomales) and Federación Andaluza de Fibromialgia y Fatiga Crónica (ALBA Andalucía). MM receiveda fellowship from Colegio Oficial de Farmacéuticos de Sevilla. This group was founded by the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER),ISCIII.Peer reviewe

Cell identity and nucleo-mitochondrial genetic context modulate OXPHOS performance and determine somatic heteroplasmy dynamics

Heteroplasmy, multiple variants of mitochondrial DNA (mtDNA) in the same cytoplasm, may be naturally generated by mutations but is counteracted by a genetic mtDNA bottleneck during oocyte development. Engineered heteroplasmic mice with nonpathological mtDNA variants reveal a nonrandom tissue-specific mtDNA segregation pattern, with few tissues that do not show segregation. The driving force for this dynamic complex pattern has remained unexplained for decades, challenging our understanding of this fundamental biological problem and hindering clinical planning for inherited diseases. Here, we demonstrate that the nonrandom mtDNA segregation is an intracellular process based on organelle selection. This cell type-specific decision arises jointly from the impact of mtDNA haplotypes on the oxidative phosphorylation (OXPHOS) system and the cell metabolic requirements and is strongly sensitive to the nuclear context and to environmental cues

A network of macrophages supports mitochondrial homeostasis in the heart

Cardiomyocytes are subjected to the intense mechanical stress and metabolic demands of the beating heart. It is unclear whether these cells, which are long-lived and rarely renew, manage to preserve homeostasis on their own. While analyzing macrophages lodged within the healthy myocardium, we discovered that they actively took up material, including mitochondria, derived from cardiomyocytes. Cardiomyocytes ejected dysfunctional mitochondria and other cargo in dedicated membranous particles reminiscent of neural exophers, through a process driven by the cardiomyocyte’s autophagy machinery that was enhanced during cardiac stress. Depletion of cardiac macrophages or deficiency in the phagocytic receptor Mertk resulted in defective elimination of mitochondria from the myocardial tissue, activation of the inflammasome, impaired autophagy, accumulation of anomalous mitochondria in cardiomyocytes, metabolic alterations, and ventricular dysfunction. Thus, we identify an immune-parenchymal pair in the murine heart that enables transfer of unfit material to preserve metabolic stability and organ function