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.

Analysis of 18F-Sodium Fluoride Positron Emission Tomography Signal Sources in Atherosclerotic Minipigs Shows Specific Binding of 18F-Sodium Fluoride to Plaque Calcifications.

Objective: 18F-sodium fluoride (18F-NaF) positron emission tomography (PET) imaging is thought to visualize active atherosclerotic plaque calcification. This is supported by the binding of 18F-NaF to plaque calcification ex vivo, but no prior studies have examined binding of 18F-NaF to human-like plaque in vivo. Our aim was to validate the specificity of 18F-NaF PET for plaque calcifications in atherosclerotic minipigs. Approach and Results: Gain-of-function PCSK9D374Y (proprotein convertase/subtilisin kexin type 9) transgenic Yucatan minipigs (n=4) were fed high-fat diet for 2.5 years to develop atherosclerosis and then subjected to 18F-NaF PET/computed tomography imaging. The heart, aorta, and iliac arteries were immediately re-scanned ex vivo after surgical extraction. Lesions from the abdominal aorta, iliac arteries, and coronary arteries were cryo-sectioned for autoradiography. Histological plaque characteristics, PET/computed tomography signal, and autoradiography were linked through regression and co-localization analysis. Arterial 18F-NaF PET signal had intensities comparable to clinical scans and colocalized moderately with calcification detected by computed tomography. Histological analysis showed calcification spanning from microcalcifications near lipid pools and necrotic core to more homogenous macrocalcifications. Comparison with arteries from autopsy cases confirmed the resemblance in localization and appearance with early human plaque calcification. Regression analysis in the abdominal aorta showed correlations with calcified plaque but could not rule out contributions from noncalcified plaque. This was resolved by autoradiography, which showed specific accumulation in plaque calcifications in all examined arteries. In the context of porcine abdominal aorta, 18F-NaF PET imaging was, however, less accurate than computed tomography for detecting small calcifications. Conclusions: 18F-NaF accumulates specifically in calcifications of atherosclerotic plaques in vivo.

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

Objective: 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. Approach and Results: 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. Conclusions: 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.

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 LmnaG609G/G609G mice ubiquitously expressing progerin. To induce progerin expression specifically in macrophages or vascular smooth muscle cells (VSMCs), we crossed Apoe-/-LmnaLCS/LCS mice with LysMCre and SM22αCre 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-/-LmnaG609G/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-/-LmnaG609G/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-/-LmnaLCS/LCSSM22αCre mice expressing progerin specifically in VSMCs, but not in Apoe-/-LmnaLCS/LCSLysMCre mice with macrophage-specific progerin expression. Moreover, Apoe-/-LmnaLCS/LCSSM22αCre 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-/-LmnaLCS/LCSSM22αCre mice, unlike Apoe-/-LmnaG609G/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.

Diabetes with poor glycaemic control does not promote atherosclerosis in genetically modified hypercholesterolaemic minipigs.

AIMS/HYPOTHESIS: Diabetes is associated with an increased risk of atherosclerotic cardiovascular disease, but whether there is a direct and independent role for impaired glucose control in atherogenesis remains uncertain. We investigated whether diabetes with poor glycaemic control would accelerate atherogenesis in a novel pig model of atherosclerosis, the D374Y-PCSK9(+) transgenic minipig. METHODS: Nineteen minipigs were fed a cholesterol-enriched, high-fat diet; ten of these pigs were injected with streptozotocin to generate a model of diabetes. Restricted feeding was implemented to control the pigs' weight gain and cholesterol intake. After 49 weeks of high-fat feeding, the major arteries were harvested for a detailed analysis of the plaque burden and histological plaque type. RESULTS: Stable hyperglycaemia was achieved in the diabetic minipigs, while the plasma total and LDL-cholesterol and creatinine levels were unaffected. Diabetes failed to increase atherosclerosis in any of the vessels examined. The plaque burden in the aorta and right coronary artery was comparable between the groups, and was even reduced in the left anterior descending (LAD) coronary and iliofemoral arteries in the diabetic pigs compared with the controls. The distribution of plaque types and the collagen and macrophage contents were similar between the groups, except for a reduced infiltration of macrophages in the LAD arteries of the diabetic pigs. CONCLUSIONS/INTERPRETATION: Poorly controlled diabetes with no alterations in plasma cholesterol or creatinine concentrations did not augment the plaque burden or promote the development of more advanced lesions in this large-animal model of human-like atherosclerosis. This is consistent with clinical studies in patients with type 1 diabetes, indicating that hyperglycaemia per se is not an independent promoter of atherosclerotic disease, but that other diabetes-associated risk factors are important.

Cardiac magnetic resonance and electroanatomical mapping of acute and chronic atrial ablation injury: a histological validation study.

AIMS: To provide a comprehensive histopathological validation of cardiac magnetic resonance (CMR) and endocardial voltage mapping of acute and chronic atrial ablation injury. METHODS AND RESULTS: 16 pigs underwent pre-ablation T2-weighted (T2W) and late gadolinium enhancement (LGE) CMR and high-density voltage mapping of the right atrium (RA) and both were repeated after intercaval linear radiofrequency ablation. Eight pigs were sacrificed following the procedure for pathological examination. A further eight pigs were recovered for 8 weeks, before chronic CMR, repeat RA voltage mapping and pathological examination. Signal intensity (SI) thresholds from 0 to 15 SD above a reference SI were used to segment the RA in CMR images and segmentations compared with real lesion volumes. The SI thresholds that best approximated histological volumes were 2.3 SD for LGE post-ablation, 14.5 SD for T2W post-ablation and 3.3 SD for LGE chronically. T2-weighted chronically always underestimated lesion volume. Acute histology showed transmural injury with coagulative necrosis. Chronic histology showed transmural fibrous scar. The mean voltage at the centre of the ablation line was 3.3 mV pre-ablation, 0.6 mV immediately post-ablation, and 0.3 mV chronically. CONCLUSION: This study presents the first histopathological validation of CMR and endocardial voltage mapping to define acute and chronic atrial ablation injury, including SI thresholds that best match histological lesion volumes. An understanding of these thresholds may allow a more informed assessment of the underlying atrial substrate immediately after ablation and before repeat catheter ablation for atrial arrhythmias.

Sphingomyelinase-responsive nanomicelles for targeting atherosclerosis.

Atherosclerosis, a leading cause of cardiovascular diseases requires approaches to enhance disease monitoring and treatment. Nanoparticles offer promising potential in this area by being customisable to target components or molecular processes within plaques, while carrying diagnostic and therapeutic agents. However, the number of biomarkers available to target this disease is limited. This study investigated the use of sphingomyelin-based nanomicelles triggered by sphingomyelinase (SMase) in atherosclerotic plaques. Accumulation of iron oxide-based nanomicelles in the plaque was demonstrated by fluorescence, MR imaging and electron microscopy. These findings demonstrate the possibility of utilising SMase as a mechanism to retain nanoprobes within plaques, thus opening up possibilities for future therapeutic interventions.

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 (PRSCAD) was calculated. The prediction was performed using combinations of PRSCAD, 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 PRSCAD-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 (PRSCAD+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: NCT02264717.

Endothelial ADAM10 controls cellular response to oxLDL and its deficiency exacerbates atherosclerosis with intraplaque hemorrhage and neovascularization in mice.

Introduction: The transmembrane protease A Disintegrin And Metalloproteinase 10 (ADAM10) displays a "pattern regulatory function," by cleaving a range of membrane-bound proteins. In endothelium, it regulates barrier function, leukocyte recruitment and angiogenesis. Previously, we showed that ADAM10 is expressed in human atherosclerotic plaques and associated with neovascularization. In this study, we aimed to determine the causal relevance of endothelial ADAM10 in murine atherosclerosis development in vivo. Methods and results: Endothelial Adam10 deficiency (Adam10 ecko ) in Western-type diet (WTD) fed mice rendered atherogenic by adeno-associated virus-mediated PCSK9 overexpression showed markedly increased atherosclerotic lesion formation. Additionally, Adam10 deficiency was associated with an increased necrotic core and concomitant reduction in plaque macrophage content. Strikingly, while intraplaque hemorrhage and neovascularization are rarely observed in aortic roots of atherosclerotic mice after 12 weeks of WTD feeding, a majority of plaques in both brachiocephalic artery and aortic root of Adam10ecko mice contained these features, suggestive of major plaque destabilization. In vitro, ADAM10 knockdown in human coronary artery endothelial cells (HCAECs) blunted the shedding of lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1) and increased endothelial inflammatory responses to oxLDL as witnessed by upregulated ICAM-1, VCAM-1, CCL5, and CXCL1 expression (which was diminished when LOX-1 was silenced) as well as activation of pro-inflammatory signaling pathways. LOX-1 shedding appeared also reduced in vivo, as soluble LOX-1 levels in plasma of Adam10ecko mice was significantly reduced compared to wildtypes. Discussion: Collectively, these results demonstrate that endothelial ADAM10 is atheroprotective, most likely by limiting oxLDL-induced inflammation besides its known role in pathological neovascularization. Our findings create novel opportunities to develop therapeutics targeting atherosclerotic plaque progression and stability, but at the same time warrant caution when considering to use ADAM10 inhibitors for therapy in other diseases.

Flanking recipient vasculature, not circulating progenitor cells, contributes to endothelium and smooth muscle in murine allograft vasculopathy.

OBJECTIVE: The prevailing view assumes that circulating endothelial and smooth muscle progenitor cells participate in allograft vasculopathy (AV), although the seminal studies in the field were not designed to distinguish between circulating and migrating cells of recipient origin. We developed a double-transplantation technique to overcome this problem and reinvestigated the origin of endothelial cells (ECs) and smooth muscle cells (SMCs) in murine AV. METHODS AND RESULTS: Carotid artery segments from BALB/c mice were allografted to apolipoprotein E(-/-) B6 mice with or without a "flanking" isograft interpositioned between the allograft and the recipient artery. Either recipient mice or interpositioned isografts expressed enhanced green fluorescent protein, and consequently, cells migrating into the allograft from the flanking vasculature could easily be tracked and distinguished from recruited circulating cells. Without immunosuppression, allograft donor cells vanished as expected, and AV developed by replacement and accumulation of ECs and SMCs of recipient origin. The double transplantation models revealed that all ECs and SMCs in AV had migrated into the allograft from the flanking vasculature without any contribution from putative progenitor cells in the blood. CONCLUSIONS: Migrating cells from the flanking vasculature, not circulating progenitor cells, are the source of recipient-derived ECs and SMCs in murine AV.

Genetic susceptibility of the arterial wall is an important determinant of atherosclerosis in C57BL/6 and FVB/N mouse strains.

OBJECTIVE: How genetic variations among inbred mouse strains translate into differences in atherosclerosis susceptibility is of significant interest for the development of new therapeutic strategies. The objective of the present study was to examine whether genetically controlled arterial wall properties influence atherosclerosis susceptibility in FVB/N (FVB) and C57BL/6 (B6) apolipoprotein E knockout (apoE(-/-)) mouse strains. METHODS AND RESULTS: Common carotid artery segments from B6 apoE(-/-), F1 apoE(-/-), and FVB apoE(-/-) mice were transplanted to hybrid F1 apoE(-/-) mice, which can accept grafts from both parental strains without adaptive immune responses. The mice were fed a high-fat diet, and atherosclerosis was induced in the transplanted artery segments by placement of a perivascular constrictive collar. Artery segments from B6 apoE(-/-) mice developed much larger atherosclerotic lesions than artery segments from FVB or F1 apoE(-/-) mice. No differences in aortic arch atherosclerosis of the recipient mice were observed between groups. CONCLUSIONS: Genetically controlled factors acting at the level of the arterial wall are important determinants of atherosclerosis susceptibility in FVB apoE(-/-) and B6 apoE(-/-) mice.

Atherosclerosis Induced by Adeno-Associated Virus Encoding Gain-of-Function PCSK9.

Induction of atherosclerosis in mice with one or more genetic alterations (e.g., conditional deletion of a gene of interest) has traditionally required crossbreeding with Apoe or Ldlr deficient mice to achieve sufficient hypercholesterolemia. However, this procedure is time consuming and generates a surplus of mice with genotypes that are irrelevant for experiments. Several alternative methods exist that obviate the need to work in mice with germline-encoded hypercholesterolemia. In this chapter, we detail an efficient and increasingly used method to induce hypercholesterolemia in mice through adeno-associated virus-mediated transfer of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene.

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

BACKGROUND: 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. OBJECTIVES: This study aimed to validate this method for plaque volume measurement in early and intermediate-advanced plaques in the carotid and femoral territories. METHODS: 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. RESULTS: 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%. CONCLUSIONS: 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.

Circulating endothelial progenitor cells do not contribute to plaque endothelium in murine atherosclerosis.

BACKGROUND: It has been reported that circulating endothelial progenitor cells (EPCs) home to and differentiate into endothelial cells after various kinds of arterial injury. By inference, EPCs are also proposed to be important in the most important arterial disease, atherosclerosis, but the evidence for this theory is not clear. In the present study, we assessed the contribution of circulating EPCs to plaque endothelium in apolipoprotein E-deficient (apoE(-/-)) mice. METHODS AND RESULTS: To investigate whether EPCs in the circulating blood are a source of plaque endothelial cells during atherogenesis, we examined plaques in lethally irradiated apoE(-/-) mice reconstituted with bone marrow cells from enhanced green fluorescent protein (eGFP) transgenic apoE(-/-) mice and plaques induced in segments of common carotid artery transplanted from apoE(-/-) mice into eGFP(+)apoE(-/-) mice. Among 4232 endothelial cells identified by a cell-type-specific marker (von Willebrand factor) and analyzed by high-resolution microscopy, we found only 1 eGFP(+). Using the Y chromosome to track cells after sex-mismatched transplants yielded similar results. To investigate whether circulating EPCs are involved in plaque reendothelialization after plaque disruption and superimposed thrombosis, we produced mechanical plaque disruptions in carotid bifurcation plaques in old lethally irradiated apoE(-/-) mice reconstituted with eGFP(+)apoE(-/-) bone marrow cells and carotid bifurcation plaques transplanted from old apoE(-/-) mice into eGFP(+)apoE(-/-) mice. Only 1 eGFP(+) endothelial cell was found among 3170 analyzed. CONCLUSIONS: Circulating EPCs rarely, if ever, contribute to plaque endothelium in apoE(-/-) mice. These findings bring into question the prevailing theory that circulating EPCs play an important role in atherogenesis.

Local Pressure Drives Low-Density Lipoprotein Accumulation and Coronary Atherosclerosis in Hypertensive Minipigs.

BACKGROUND: The mechanisms by which hypertension accelerates coronary artery disease are poorly understood. Patients with hypertension often have confounding humoral changes, and to date, no experimental models have allowed analysis of the isolated effect of pressure on atherosclerosis in a setting that recapitulates the dimensions and biomechanics of human coronary arteries. OBJECTIVES: This study sought to analyze the effect of pressure on coronary atherosclerosis and explore the underlying mechanisms. METHODS: Using inflatable suprarenal aortic cuffs, we increased mean arterial pressure by >30 mm Hg in the cephalad body part of wild-type and hypercholesterolemic proprotein convertase subtilisin kexin type 9 (PCSK9)D374Y Yucatan minipigs for >1 year. Caudal pressures remained normal. RESULTS: Under hypercholesterolemic conditions in PCSK9D374Y transgenic minipigs, cephalad hypertension accelerated coronary atherosclerosis to almost 5-fold with consistent development of fibroatheromas that were sufficiently large to cause stenosis on computed tomography angiography. This was caused by local pressure forces, because vascular beds shielded from hypertension, but exposed to the same humoral factors, showed no changes in lesion formation. The same experiment was conducted under normocholesterolemic conditions in wild-type minipigs to examine the underlying mechanisms. Hypertension produced clear changes in the arterial proteome with increased abundance of mechanical strength proteins and reduced levels of infiltrating plasma macromolecules. This was paralleled by increased smooth muscle cells and increased intimal accumulation of low-density lipoproteins in the coronary arteries. CONCLUSIONS: Increased pressure per se facilitates coronary atherosclerosis. Our data indicate that restructuring of the artery to match increased tensile forces in hypertension alters the passage of macromolecules and leads to increased intimal accumulation of low-density lipoproteins.

Effects of castration on atherosclerosis in Yucatan minipigs with genetic hypercholesterolemia.

BACKGROUND: Low plasma testosterone, either spontaneous or as a result of androgen deprivation therapy for prostate cancer, is associated with an increased risk of cardiovascular events. The underlying mechanism in humans is not understood. Experimental studies in mice have shown that castration facilitates atherogenesis and may increase signs of plaque vulnerability. Pigs used for translational atherosclerosis research have frequently been castrated for practical or commercial reasons, but the effect of castration on atherosclerosis has never been systematically evaluated in pigs. OBJECTIVE: To study the effect of castration on atherosclerotic plaque burden and type in genetically modified minipigs with hypercholesterolemia. METHODS: Newborn male Yucatan minipigs with transgenic overexpression of a human gain-of-function mutant of proprotein convertase subtilisin/kexin type 9 were randomized to undergo orchiectomy (n = 8) or serve as controls (n = 6). Minipigs were started on high-fat diet at 3 months of age and the amount and composition of atherosclerotic lesions were analyzed at 12 months of age. Plasma lipid profiles and behavioral parameters were also assessed. RESULTS: Plasma lipids were slightly affected to a more atherogenic profile by orchiectomy, but atherosclerotic lesion size was unaltered in the LAD, thoracic aorta, abdominal aorta, and iliac arteries. The distribution of lesion types (xanthomas, pathological intimal thickening and fibroatheromas) were also not statistically different between groups in any of the examined vascular territories. The abdominal aorta developed the most advanced stages of disease with reproducible fibroatheroma formation, and here it was found that the area of necrotic core was significantly increased in orchiectomized pigs compared with controls. Orchiectomy also reduced aggressive behavior. CONCLUSIONS: Castration does not alter the burden of atherosclerosis in hypercholesterolemic Yucatan minipigs, but may increase necrotic core area in fibroatheromas.

Tissue volume and activity mapping using total intensity projection of PET/CT images.

Autoradiography using phosphor imaging screens is often used to characterize tissue distribution of positron emission tomography (PET) radiotracers. PET tracers emit positrons with limited penetration range, and valid quantitative autoradiography can therefore only be achieved in thin tissue slices. However, in some settings, quantitative tracer profiling in thick tissues is required. Our aim was to develop a reliable method for this purpose. In this paper, we present a method based on total intensity projections (TIPs) of PET and computed tomography (CT) images. We show theoretically and experimentally that tissue total activity and tissue volume maps can be derived from the TIPs of PET and CT images, respectively. We also show that these maps are free of signal displacement artifacts in the direction of projection. To demonstrate the utility of the approach, we obtain and compare TIP-based maps and autoradiography of ex-vivo atherosclerotic minipig aortas following in-vivo injection of 18F-fluorodeoxyglucose. We show that autoradiography of the thick aortas yields distorted results due to positron range effects, whereas TIP-mapping is free from such bias. The TIP-based maps may, thus, provide a low-resolution alternative to autoradiography, when tracer accumulation profiling in thick tissues is required.