Oil Red O and Hematoxylin and Eosin Staining for Quantification of Atherosclerosis Burden in Mouse Aorta and Aortic Root

Methods for staining tissues with Oil Red O and hematoxylin-eosin are classical histological techniques that are widely used to quantify atherosclerotic burden in mouse tissues because of their ease of use, reliability, and the large amount of information they provide. These stains can provide quantitative data about the impact of a genetic or environmental factor on atherosclerotic burden and on the initiation, progression, or regression of the disease, and can also be used to evaluate the efficacy of drugs designed to prevent or treat atherosclerosis. This chapter provides protocols for quantifying atherosclerotic burden in mouse aorta and aortic root, including methods for dissection, Oil Red O staining, hematoxylin-eosin staining, and image analysis.The authors thank Simon Bartlett for editorial assistance. Work in VA’s laboratory is supported by grants SAF2013-46663-R and RD12/0042/0028 from the Spanish Ministerio de Economía y Competitividad (MINECO) with co-funding from the Fondo Europeo de Desarrollo Regional (FEDER), the European Commission (Liphos, Grant Agreement No. 317916), and the Progeria Research Foundation (Established Investigator Award). The CNIC is supported by the MINECO and the Pro-CNIC Foundation

Vascular Smooth Muscle Cell-Specific Progerin Expression Provokes Contractile Impairment in a Mouse Model of Hutchinson-Gilford Progeria Syndrome that Is Ameliorated by Nitrite Treatment

Cardiovascular disease (CVD) is the main cause of death worldwide, and aging is its leading risk factor. Aging is much accelerated in Hutchinson-Gilford progeria syndrome (HGPS), an ultra-rare genetic disorder provoked by the ubiquitous expression of a mutant protein called progerin. HGPS patients die in their teens, primarily due to cardiovascular complications. The primary causes of age-associated CVD are endothelial dysfunction and dysregulated vascular tone; however, their contribution to progerin-induced CVD remains poorly characterized. In the present study, we found that progeroid LmnaG609G/G609G mice with ubiquitous progerin expression show both endothelial dysfunction and severe contractile impairment. To assess the relative contribution of specific vascular cell types to these anomalies, we examined LmnaLCS/LCSTie2Cretg/+ and LmnaLCS/LCSSm22αCretg/+ mice, which express progerin specifically in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. Whereas vessel contraction was impaired in mice with VSMC-specific progerin expression, we observed no endothelial dysfunction in mice with progerin expression restricted to VSMCs or ECs. Vascular tone regulation in progeroid mice was ameliorated by dietary sodium nitrite supplementation. Our results identify VSMCs as the main cell type causing contractile impairment in a mouse model of HGPS that is ameliorated by nitrite treatment.Work in V.A.’s lab was supported by the Spanish Ministerio de Ciencia e Innovación (MCI, grant SAF2016-79490-R), with co-funding from the European Regional Development Fund (ERDF, “Una manera de hacer Europa”). L.d.C. was supported by a Jordi Soler postdoctoral contract from the Red de Investigación Cardiovascular (RETIC Program, Instituto de Salud Carlos III), and A.S.-L. was supported by a predoctoral contract from the MCI (SVP-2014-068334) and by a grant from Asociación Apadrina la Ciencia-Ford España-Ford Motor Company Fund. The CNIC is supported by the MCI, the Instituto de Salud Carlos III, and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant SEV-2015-0505).S

Atherosclerotic plaque development in mice is enhanced by myeloid ZEB1 downregulation.

Accumulation of lipid-laden macrophages within the arterial neointima is a critical step in atherosclerotic plaque formation. Here, we show that reduced levels of the cellular plasticity factor ZEB1 in macrophages increase atherosclerotic plaque formation and the chance of cardiovascular events. Compared to control counterparts (Zeb1WT/ApoeKO), male mice with Zeb1 ablation in their myeloid cells (Zeb1∆M/ApoeKO) have larger atherosclerotic plaques and higher lipid accumulation in their macrophages due to delayed lipid traffic and deficient cholesterol efflux. Zeb1∆M/ApoeKO mice display more pronounced systemic metabolic alterations than Zeb1WT/ApoeKO mice, with higher serum levels of low-density lipoproteins and inflammatory cytokines and larger ectopic fat deposits. Higher lipid accumulation in Zeb1∆M macrophages is reverted by the exogenous expression of Zeb1 through macrophage-targeted nanoparticles. In vivo administration of these nanoparticles reduces atherosclerotic plaque formation in Zeb1∆M/ApoeKO mice. Finally, low ZEB1 expression in human endarterectomies is associated with plaque rupture and cardiovascular events. These results set ZEB1 in macrophages as a potential target in the treatment of atherosclerosis.S

Generation and characterization of a novel knockin minipig model of Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder for which no cure exists. The disease is characterized by premature aging and inevitable death in adolescence due to cardiovascular complications. Most HGPS patients carry a heterozygous de novo LMNA c.1824C > T mutation, which provokes the expression of a dominant-negative mutant protein called progerin. Therapies proven effective in HGPS-like mouse models have yielded only modest benefit in HGPS clinical trials. To overcome the gap between HGPS mouse models and patients, we have generated by CRISPR-Cas9 gene editing the first large animal model for HGPS, a knockin heterozygous LMNA c.1824C > T Yucatan minipig. Like HGPS patients, HGPS minipigs endogenously co-express progerin and normal lamin A/C, and exhibit severe growth retardation, lipodystrophy, skin and bone alterations, cardiovascular disease, and die around puberty. Remarkably, the HGPS minipigs recapitulate critical cardiovascular alterations seen in patients, such as left ventricular diastolic dysfunction, altered cardiac electrical activity, and loss of vascular smooth muscle cells. Our analysis also revealed reduced myocardial perfusion due to microvascular damage and myocardial interstitial fibrosis, previously undescribed readouts potentially useful for monitoring disease progression in patients. The HGPS minipigs provide an appropriate preclinical model in which to test human-size interventional devices and optimize candidate therapies before advancing to clinical trials, thus accelerating the development of effective applications for HGPS patients.This project was mainly supported by an Established Investigator Award from the Progeria Research Foundation (2014-52), and from the Spanish Ministerio de Ciencia, Innovación y Universidades (MCIU), and the European Regional Development Fund (FEDER, “A way to build Europe”) (SAF2016-79490-R, CB16/11/00405). Ana Barettino has a predoctoral contract from MCIU (BES-2017-079705). Work at Universidad de Murcia is supported by Fundación Seneca-Agencia de Ciencia y Tecnología de la Región de Murcia (20040/GERM/16). The CNIC is supported by the MCIU and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Search for light bosons in decays of the 125 GeV Higgs boson in proton-proton collisions at root s=8 TeV

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Search for new physics with dijet angular distributions in proton-proton collisions at root S = 13 TeV

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Observation of Charge-Dependent Azimuthal Correlations in p-Pb Collisions and Its Implication for the Search for the Chiral Magnetic Effect

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Cross section measurement of t-channel single top quark production in pp collisions at root s=13 TeV

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Search for single production of vector-like quarks decaying into a b quark and a W boson in proton-proton collisions at root s=13 TeV

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Search for dijet resonances in proton-proton collisions at root s=13 TeV and constraints on dark matter and other models

Correction: DOI:10.1016/j.physletb.2017.09.029Peer reviewe