Vascular Smooth Muscle-Specific Progerin Expression Accelerates Atherosclerosis and Death in a Mouse Model of Hutchinson-Gilford Progeria Syndrome

Background: Progerin, an aberrant protein that accumulates with age, causes the rare genetic disease Hutchinson-Gilford progeria syndrome (HGPS). Patients who have HGPS exhibit ubiquitous progerin expression, accelerated aging and atherosclerosis, and die in their early teens, mainly of myocardial infarction or stroke. The mechanisms underlying progerin-induced atherosclerosis remain unexplored, in part, because of the lack of appropriate animal models. Methods: We generated an atherosclerosis-prone model of HGPS by crossing apolipoprotein E-deficient (Apoe(-/-)) mice with Lmna(G609G/G609G) mice ubiquitously expressing progerin. To induce progerin expression specifically in macrophages or vascular smooth muscle cells (VSMCs), we crossed Apoe(-/-)Lmna(LCS/LCS) mice with LysMCre and SM22Cre mice, respectively. Progerin expression was evaluated by polymerase chain reaction and immunofluorescence. Cardiovascular alterations were determined by immunofluorescence and histology in male mice fed normal chow or a high-fat diet. In vivo low-density lipoprotein retention was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein. Cardiac electric defects were evaluated by electrocardiography. Results:Apoe(-/-)Lmna(G609G/G609G) mice with ubiquitous progerin expression exhibited a premature aging phenotype that included failure to thrive and shortened survival. In addition, high-fat diet-fed Apoe(-/-)Lmna(G609G/G609G) mice developed a severe vascular pathology, including medial VSMC loss and lipid retention, adventitial fibrosis, and accelerated atherosclerosis, thus resembling most aspects of cardiovascular disease observed in patients with HGPS. The same vascular alterations were also observed in Apoe(-/-)Lmna(LCS/LCS)SM22Cre mice expressing progerin specifically in VSMCs, but not in Apoe(-/-)Lmna(LCS/LCS)LysMCre mice with macrophage-specific progerin expression. Moreover, Apoe(-/-)Lmna(LCS/LCS)SM22Cre mice had a shortened lifespan despite the lack of any overt aging phenotype. Aortas of ubiquitously and VSMC-specific progerin-expressing mice exhibited increased retention of fluorescently labeled human low-density lipoprotein, and atheromata in both models showed vulnerable plaque features. Immunohistopathological examination indicated that Apoe(-/-)Lmna(LCS/LCS)SM22Cre mice, unlike Apoe(-/-)Lmna(G609G/G609G) mice, die of atherosclerosis-related causes. Conclusions: We have generated the first mouse model of progerin-induced atherosclerosis acceleration, and demonstrate that restricting progerin expression to VSMCs is sufficient to accelerate atherosclerosis, trigger plaque vulnerability, and reduce lifespan. Our results identify progerin-induced VSMC death as a major factor triggering atherosclerosis and premature death in HGPS.Work in Dr Andres' laboratory is supported by grants from the Spanish Ministerio de Economia, Industria y Competitividad (MEIC) (SAF2016-79490-R) and the Instituto de Salud Carlos III (AC16/00091, AC17/00067) with co-funding from the Fondo Europeo de Desarrollo Regional (FEDER, ``Una manera de hacer Europa´´), the Progeria Research Foundation (Established Investigator Award 2014-52), and the Fundacio Marato TV3 (122/C/2015). The MEIC supported Dr Hamczyk (´´Formacion de Personal Investigador´´ predoctoral contract BES-2011-043938) and Dr Villa-Bellosta (´´Juan de la Cierva´´ JCI-2011-09663 postdoctoral contract). The Instituto Universitario de Oncologia is supported by Obra Social Cajastur. The Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) is supported by the MEIC and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (award SEV-2015-0505).S

Differences in Hypercholesterolemia and Atherogenesis Induced by Common Androgen Deprivation Therapies in Male Mice

Background-Treatment of prostate cancer often involves androgen deprivation therapy (ADT) by gonadotropin-releasing hormone (GnRH) receptor agonists, GnRH receptor antagonists, or orchiectomy. ADT may increase the rate of cardiovascular disease events, but recent clinical studies suggested that not all means of ADT carry the same risk, raising the possibility of non-testosterone-mediated effects of different forms of ADT on atherosclerosis. Here we compared effects of ADT on atherosclerosis in intact and orchiectomized Apoe-deficient mice. Methods and Results-Chow-fed Apoe-deficient mice were allocated to orchiectomy and/or monthly injections with the GnRH receptor agonist leuprolide or the GnRH receptor antagonist degarelix. Atherosclerosis was quantified at 26 weeks of age in the aortic arch by en face examination and in the aortic root by histology. In intact Apoe-deficient mice, all types of ADT reduced testosterone production to castration levels. Although hypercholesterolemia was accentuated in leuprolide-treated mice, the amount and composition of atherosclerosis was not different between the different types of ADT. In orchiectomized Apoe-deficient mice, leuprolide, but not degarelix, augmented hypercholesterolemia, changed body, thymus, and spleen weights, and increased atherosclerosis in the aortic root. No direct effects of the drugs were detectable on cytokine secretion from murine bone marrow-derived macrophages or on splenocyte proliferation. Conclusions-No differences in the development of atherosclerosis were detected among groups of intact Apoe-deficient mice treated with different types of ADT. A pro-atherogenic, possibly cholesterol-mediated, effect of leuprolide was seen in orchiectomized mice that might be relevant for understanding the potential cardiovascular risk associated with GnRH agonist-based ADT.The study was investigator-initiated and sponsored by Ferring Pharmaceuticals.S

High-fructose feeding does not induce steatosis or non-alcoholic fatty liver disease in pigs

Non-alcoholic fatty liver disease (NAFLD) is an increasingly prevalent condition that has been linked to high-fructose corn syrup consumption with induction of hepatic de novo lipogenesis (DNL) as the suggested central mechanism. Feeding diets very high in fructose (> 60%) rapidly induce several features of NAFLD in rodents, but similar diets have not yet been applied in larger animals, such as pigs. With the aim to develop a large animal NAFLD model, we analysed the effects of feeding a high-fructose (HF, 60% w/w) diet for four weeks to castrated male Danish Landrace-York-Duroc pigs. HF feeding upregulated expression of hepatic DNL proteins, but levels were low compared with adipose tissue. No steatosis or hepatocellular ballooning was seen on histopathological examination, and plasma levels of transaminases were similar between groups. Inflammatory infiltrates and the amount of connective tissue was slightly elevated in liver sections from fructose-fed pigs, which was corroborated by up-regulation of macrophage marker expression in liver homogenates. Supported by RNA-profiling, quantitative protein analysis, histopathological examination, and biochemistry, our data suggest that pigs, contrary to rodents and humans, are protected against fructose-induced steatosis by relying on adipose tissue rather than liver for DNL.The study was supported by a Synergy Grant from the Novo Nordisk Foundation (NNF14OC0011537) and by the Danish National Resarch Foundation (ATLAS center). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Capacity for LDL (Low-Density Lipoprotein) Retention Predicts the Course of Atherogenesis in the Murine Aortic Arch.

BACKGROUND To cause atherosclerosis, LDLs (low-density lipoproteins) must first pass through the endothelium and then become retained in the arterial matrix. Which of these two processes is rate-limiting and predicts the topography of plaque formation remains controversial. To investigate this issue, we performed high-resolution mapping of LDL entry and retention in murine aortic arches before and during atherosclerosis development. METHODS Maps of LDL entry and retention were created by injecting fluorescently labeled LDL followed by near-infrared scanning and whole-mount confocal microscopy after 1 hour (entry) and 18 hours (retention). By comparing arches between normal mice and mice with short-term hypercholesterolemia, we analyzed changes in LDL entry and retention during the LDL accumulation phase that precedes plaque formation. Experiments were designed to secure equal plasma clearance of labeled LDL in both conditions. RESULTS We found that LDL retention is the overall limiting factor for LDL accumulation but that the capacity for LDL retention varied substantially over surprisingly short distances. The inner curvature region, previously considered a homogenous atherosclerosis-prone region, consisted of dorsal and ventral zones with high capacity and a central zone with low capacity for continued LDL retention. These features predicted the temporal pattern of atherosclerosis, which first appeared in the border zones and later in the central zone. The limit to LDL retention in the central zone was intrinsic to the arterial wall, possibly caused by saturation of the binding mechanism, and was lost upon conversion to atherosclerotic lesions. CONCLUSIONS Capacity for continued LDL retention varies over short distances and predicts where and when atherosclerosis develops in the mouse aortic arch.This study was supported by grants from the Ministerio de Economía, Industria y Competitividad (MEIC) with cofunding from the European Regional Development Fund (SAF2016-75580-R and PID2019-108568RB-I00), the Novo Nordisk Foundation (NNF17OC0030688) and the La Caixa Health Research Programme (HR20-00075, AtheroConvergence). V. Labrador-Cantarero is supported by FEDER “Una manera de hacer Europa” for the project In Vivo Advanced Nanoscopy at the ICTS–ReDib–TRIMA–CNIC. CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

Fibrous Caps in Atherosclerosis Form by Notch-Dependent Mechanisms Common to Arterial Media Development.

Atheromatous fibrous caps are produced by smooth muscle cells (SMCs) that are recruited to the subendothelial space. We tested whether the recruitment mechanisms are the same as in embryonic artery development, which relies prominently on Notch signaling to form the subendothelial medial SMC layers. Notch elements were expressed in regions of fibrous cap in human and mouse plaques. To assess the causal role of Notch signaling in cap formation, we studied atherosclerosis in mice where the Notch pathway was inactivated in SMCs by conditional knockout of the essential effector transcription factor RBPJ (recombination signal-binding protein for immunoglobulin kappa J region). The recruitment of cap SMCs was significantly reduced without major effects on plaque size. Lineage tracing revealed the accumulation of SMC-derived plaque cells in the cap region was unaltered but that Notch-defective cells failed to re-acquire the SMC phenotype in the cap. Conversely, to analyze whether the loss of Notch signaling is required for SMC-derived cells to accumulate in atherogenesis, we studied atherosclerosis in mice with constitutive activation of Notch signaling in SMCs achieved by conditional expression of the Notch intracellular domain. Forced Notch signaling inhibited the ability of medial SMCs to contribute to plaque cells, including both cap SMCs and osteochondrogenic cells, and significantly reduced atherosclerosis development. Sequential loss and gain of Notch signaling is needed to build the cap SMC population. The shared mechanisms with embryonic arterial media assembly suggest that the cap forms as a neo-media that restores the connection between endothelium and subendothelial SMCs, transiently disrupted in early atherogenesis.This study was supported by a grant from the Ministerio de Ciencia e Innovación with cofunding from the European Regional Development Fund (SAF2016- 75580-R and PID2019-108568RB-I00 to J.F. Bentzon and SAF2016-78370-R to J.L. de la Pompa) and from the Novo Nordisk Foundation (NNF17OC0030688 to. J.F. Bentzon). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

18Fluorodeoxyglucose Accumulation in Arterial Tissues Determined by PET Signal Analysis

BACKGROUND: Arterial 18fluorodeoxyglucose (FDG) positron emission tomography (PET) is considered a measure of atherosclerotic plaque macrophages and is used for quantification of disease activity in clinical trials, but the distribution profile of FDG across macrophages and other arterial cells has not been fully clarified. OBJECTIVES: The purpose of this study was to analyze FDG uptake in different arterial tissues and their contribution to PET signal in normal and atherosclerotic arteries. METHODS: Wild-type and D374Y-PCSK9 transgenic Yucatan minipigs were fed a high-fat, high-cholesterol diet to induce atherosclerosis and subjected to a clinical FDG-PET and computed tomography scan protocol. Volumes of arterial media, intima/lesion, macrophage-rich, and hypoxic tissues were measured in serial histological sections. Distributions of FDG in macrophages and other arterial tissues were quantified using modeling of the in vivo PET signal. In separate transgenic minipigs, the intra-arterial localization of FDG was determined directly by autoradiography. RESULTS: Arterial FDG-PET signal appearance and intensity were similar to human imaging. The modeling approach showed high accuracy in describing the FDG-PET signal and revealed comparable FDG accumulation in macrophages and other arterial tissues, including medial smooth muscle cells. These findings were verified directly by autoradiography of normal and atherosclerotic arteries. CONCLUSIONS: FDG is taken up comparably in macrophage-rich and -poor arterial tissues in minipigs. This offers a mechanistic explanation to a growing number of observations in clinical imaging studies that have been difficult to reconcile with macrophage-selective FDG uptake.This study was supported by the Danish Council for Independent Research/Medical Sciences, Lundbeck Foundation, Danish Heart Foundation, and Aarhus University Research Foundation (AU IDEAS). The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades, and the Pro CNIC Foundation; and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Dr. Bentzon has served as a consultant for Novo Nordisk A/S; and has within the last 5 years received an investigator-initiated preclinical research grant from Regeneron PharmaceuticalsS

Combining Polygenic and Proteomic Risk Scores With Clinical Risk Factors to Improve Performance for Diagnosing Absence of Coronary Artery Disease in Patients With De Novo Chest Pain

Background: Patients with de novo chest pain, referred for evaluation of possible coronary artery disease (CAD), frequently have an absence of CAD resulting in millions of tests not having any clinical impact. The objective of this study was to investigate whether polygenic risk scores and targeted proteomics improve the prediction of absence of CAD in patients with suspected CAD, when added to the PROMISE (Prospective Multicenter Imaging Study for Evaluation of Chest Pain) minimal risk score (PMRS). Methods: Genotyping and targeted plasma proteomics (N=368 proteins) were performed in 1440 patients with symptoms suspected to be caused by CAD undergoing coronary computed tomography angiography. Based on individual genotypes, a polygenic risk score for CAD (PRS CAD ) was calculated. The prediction was performed using combinations of PRS CAD , proteins, and PMRS as features in models using stability selection and machine learning. Results: Prediction of absence of CAD yielded an area under the curve of PRS CAD -model, 0.64±0.03; proteomic-model, 0.58±0.03; and PMRS model, 0.76±0.02. No significant correlation was found between the genetic and proteomic risk scores (Pearson correlation coefficient, −0.04; P =0.13). Optimal predictive ability was achieved by the full model (PRS CAD +protein+PMRS) yielding an area under the curve of 0.80±0.02 for absence of CAD, significantly better than the PMRS model alone ( P <0.001). For reclassification purpose, the full model enabled down-classification of 49% (324 of 661) of the 5% to 15% pretest probability patients and 18% (113 of 611) of >15% pretest probability patients. Conclusions: For patients with chest pain and low-intermediate CAD risk, incorporating targeted proteomics and polygenic risk scores into the risk assessment substantially improved the ability to predict the absence of CAD. Genetics and proteomics seem to add complementary information to the clinical risk factors and improve risk stratification in this large patient group. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT0226471

New 3-Dimensional Volumetric Ultrasound Method for Accurate Quantification of Atherosclerotic Plaque Volume.

Carotid and femoral plaque burden is a recognized biomarker of cardiovascular disease risk. A new electronic-sweep 3-dimensional (3D)-matrix transducer method can improve the functionality and image quality of vascular ultrasound atherosclerosis imaging. This study aimed to validate this method for plaque volume measurement in early and intermediate-advanced plaques in the carotid and femoral territories. Plaque volumes were measured ex vivo in pig carotid and femoral artery specimens by 3-dimensional vascular ultrasound (3DVUS) using a 3D-matrix (electronic-sweep) transducer and its associated 3D plaque quantification software, and were compared with gold-standard histology. To test the clinical feasibility and accuracy of the 3D-matrix transducer, an experiment was conducted in intermediate-high risk individuals with carotid and femoral atherosclerosis. The results were compared with those obtained using the previously validated mechanical-sweep 3D transducer and established 2-dimensional (2D)-based plaque quantification software. In the ex vivo study, the authors assessed 19 atherosclerotic plaques (plaque volume, 0.76 µL-56.30 μL), finding strong agreement between measurements with the 3D-matrix transducer and the histological gold-standard (intraclass correlation coefficient [ICC]: 0.992; [95% CI: 0.978-0.997]). In the clinical analysis of 20 patients (mean age 74.6 ± 4.45 years; 40% men), the authors found 64 (36 carotid and 28 femoral) of 80 scanned territories with atherosclerosis (measured atherosclerotic volume, 10 μL-859 μL). There was strong agreement between measurements made from electronic-sweep and mechanical-sweep 3DVUS transducers (ICC: 0.997 [95% CI: 0.995-0.998]). Agreement was also high between plaque volumes estimated by the 2D and 3D plaque quantification software applications (ICC: 0.999 [95% CI: 0.998-0.999]). Analysis time was significantly shorter with the 3D plaque quantification software than with the 2D multislice approach with a mean time reduction of 46%. 3DVUS using new matrix transducer technology, together with improved 3D plaque quantification software, simplifies the accurate volume measurement of early (small) and intermediate-advanced plaques located in carotid and femoral arteries.This study was partially funded by grants from the Ministerio de Economia, Industria y Competividad (MEIC) with cofunding from the European Regional Development Fund (ERDF) (SAF2016-75580-R to Dr Bentzon) and (BES-2016-076633 to Dr Nogales). Research funding was also received from the Instituto de Salud Carlos III Spain (PIE16/ 00021 to Drs Bueno and Fuster). The CNIC is supported by the Min- isterio de Ciencia, Innovacion y Universidades (MICINN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020-001041-S). This study forms part of a Master Research Agreement between the CNIC and Philips Healthcare. Drs Sánchez- González, Entrekin, and Collet-Billon are employees of Philips Healthcare. All other authors have reported that they have no re- lationships to disclose related to the contents of this paper.S

Cholesterol lowering depletes atherosclerotic lesions of smooth muscle cell-derived fibromyocytes and chondromyocytes

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