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Currently, there is a limited ability to interactively study developmental cardiac mechanics and physiology. We therefore combined light-sheet fluorescence microscopy (LSFM) with virtual reality (VR) to provide a hybrid platform for 3D architecture and time-dependent cardiac contractile function characterization. By taking advantage of the rapid acquisition, high axial resolution, low phototoxicity, and high fidelity in 3D and 4D (3D spatial + 1D time or spectra), this VR-LSFM hybrid methodology enables interactive visualization and quantification otherwise not available by conventional methods, such as routine optical microscopes. We hereby demonstrate multiscale applicability of VR-LSFM to (a) interrogate skin fibroblasts interacting with a hyaluronic acid–based hydrogel, (b) navigate through the endocardial trabecular network during zebrafish development, and (c) localize gene therapy-mediated potassium channel expression in adult murine hearts. We further combined our batch intensity normalized segmentation algorithm with deformable image registration to interface a VR environment with imaging computation for the analysis of cardiac contraction. Thus, the VR-LSFM hybrid platform demonstrates an efficient and robust framework for creating a user-directed microenvironment in which we uncovered developmental cardiac mechanics and physiology with high spatiotemporal resolution.A user-directed perspective for studying cardiac mechanics, physiology, and developmental biology at single-cell resolution.

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I-VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

The advent of 4-dimensional (4D) light-sheet fluorescence microscopy (LSFM) has provided an entry point for rapid image acquisition to uncover real-time cardiovascular structure and function with high axial resolution and minimal photo-bleaching/-toxicity. We hereby review the fundamental principles of our LSFM system to investigate cardiovascular morphogenesis and regeneration after injury. LSFM enables us to reveal the micro-circulation of blood cells in the zebrafish embryo and assess cardiac ventricular remodeling in response to chemotherapy-induced injury using an automated segmentation approach. Next, we review two distinct mechanisms underlying zebrafish vascular regeneration following tail amputation. We elucidate the role of endothelial Notch signaling to restore vascular regeneration after exposure to the redox active ultrafine particles (UFP) in air pollutants. By manipulating the blood viscosity and subsequently, endothelial wall shear stress, we demonstrate the mechanism whereby hemodynamic shear forces impart both mechanical and metabolic effects to modulate vascular regeneration. Overall, the implementation of 4D LSFM allows for the elucidation of mechanisms governing cardiovascular injury and regeneration with high spatiotemporal resolution.

AimsRedox active ultrafine particles (UFP, d

This study sought to develop an automated segmentation approach based on histogram analysis of raw axial images acquired by light-sheet fluorescent imaging (LSFI) to establish rapid reconstruction of the 3-D zebrafish cardiac architecture in response to doxorubicin-induced injury and repair. Input images underwent a 4-step automated image segmentation process consisting of stationary noise removal, histogram equalization, adaptive thresholding, and image fusion followed by 3-D reconstruction. We applied this method to 3-month old zebrafish injected intraperitoneally with doxorubicin followed by LSFI at 3, 30, and 60 days post-injection. We observed an initial decrease in myocardial and endocardial cavity volumes at day 3, followed by ventricular remodeling at day 30, and recovery at day 60 (P P P

We designed a novel 6-point electrochemical impedance spectroscopy (EIS) sensor with 15 combinations of permutations for the 3-D mapping and detection of metabolically active atherosclerotic lesions. Two rows of 3 stretchable electrodes circumferentially separated by 120° were mounted on an inflatable balloon for intravascular deployment and endoluminal interrogation. The configuration and 15 permutations of 2-point EIS electrodes allowed for deep arterial penetration via alternating current (AC) to detect varying degrees of lipid burden with distinct impedance profiles (Ω). By virtue of the distinctive impedimetric signature of metabolically active atherosclerotic lesions, a detailed impedance map was acquired, with the 15 EIS permutations uncovering early stages of disease characterized by fatty streak lipid accumulation in the New Zealand White rabbit model of atherosclerosis. Both the equivalent circuit and statistical analyses corroborated the 3-D EIS permutations to detect small, angiographically invisible, lipid-rich lesions, with translational implications for early atherosclerotic disease detection and prevention of acute coronary syndromes or strokes.

Zebrafish are increasingly utilized to model cardiomyopathies and regeneration. Current methods evaluating cardiac function have known limitations, fail to reliably detect focal mechanics, and are not readily feasible in zebrafish. We developed a semiautomated, open-source method - displacement analysis of myocardial mechanical deformation (DIAMOND) - for quantitative assessment of 4D segmental cardiac function. We imaged transgenic embryonic zebrafish in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Our method permits the derivation of a transformation matrix to quantify the time-dependent 3D displacement of segmental myocardial mass centroids. Through treatment with doxorubicin, and by chemically and genetically manipulating the myocardial injury-activated Notch signaling pathway, we used DIAMOND to demonstrate that basal ventricular segments adjacent to the atrioventricular canal display the highest 3D displacement and are also the most susceptible to doxorubicin-induced injury. Thus, DIAMOND provides biomechanical insights into in vivo segmental cardiac function scalable to high-throughput research applications.

The adult zebrafish is an emerging vertebrate model for studying human cardiomyopathies; however, whether the simple zebrafish heart can model different subtypes of cardiomyopathies, such as dilated cardiomyopathy (DCM), remains elusive. Here, we generated and characterized an inherited DCM model in adult zebrafish and used this model to search for therapeutic strategies. We employed transcription activator-like effector nuclease (TALEN) genome editing technology to generate frame-shift mutants for the zebrafish ortholog of human BCL2-associated athanogene 3 (BAG3), an established DCM-causative gene. As in mammals, the zebrafish bag3 homozygous mutant (bag3e2/e2) exhibited aberrant proteostasis, as indicated by impaired autophagy flux and elevated ubiquitinated protein aggregation. Through comprehensive phenotyping analysis of the mutant, we identified phenotypic traits that resembled DCM phenotypes in mammals, including cardiac chamber enlargement, reduced ejection fraction characterized by increased end-systolic volume/body weight (ESV/BW), and reduced contractile myofibril activation kinetics. Nonbiased transcriptome analysis identified the hyperactivation of the mechanistic target of rapamycin (mTOR) signaling in bag3e2/e2 mutant hearts. Further genetic studies showed that mtorxu015/+, an mTOR haploinsufficiency mutant, repaired abnormal proteostasis, improved cardiac function and rescued the survival of the bag3e2/e2 mutant. This study established the bag3e2/e2 mutant as a DCM model in adult zebrafish and suggested mtor as a candidate therapeutic target gene for BAG3 cardiomyopathy., Summary: This study shows that adult bag3 knockout mutant zebrafish can be used as a model for DCM, and haploinsufficiency of mTOR is cardioprotective.

While virtual reality (VR) has potential in enhancing cardiovascular diagnosis and treatment, prerequisite labor-intensive image segmentation remains an obstacle for seamlessly simulating 4-dimensional (4-D, 3-D + time) imaging data in an immersive, physiological VR environment. We applied deformable image registration (DIR) in conjunction with 3-D reconstruction and VR implementation to recapitulate developmental cardiac contractile function from light-sheet fluorescence microscopy (LSFM). This method addressed inconsistencies that would arise from independent segmentations of time-dependent data, thereby enabling the creation of a VR environment that fluently simulates cardiac morphological changes. By analyzing myocardial deformation at high spatiotemporal resolution, we interfaced quantitative computations with 4-D VR. We demonstrated that our LSFM-captured images, followed by DIR, yielded average dice similarity coefficients of 0.92 ± 0.05 (n = 510) and 0.93 ± 0.06 (n = 240) when compared to ground truth images obtained from Otsu thresholding and manual segmentation, respectively. The resulting VR environment simulates a wide-angle zoomed-in view of motion in live embryonic zebrafish hearts, in which the cardiac chambers are undergoing structural deformation throughout the cardiac cycle. Thus, this technique allows for an interactive micro-scale VR visualization of developmental cardiac morphology to enable high resolution simulation for both basic and clinical science.

The ability to image tissue morphogenesis in real-time and in 3-dimensions (3-D) remains an optical challenge. The advent of light-sheet fluorescence microscopy (LSFM) has advanced developmental biology and tissue regeneration research. In this review, we introduce a LSFM system in which the illumination lens reshapes a thin light-sheet to rapidly scan across a sample of interest while the detection lens orthogonally collects the imaging data. This multiscale strategy provides deep-tissue penetration, high-spatiotemporal resolution, and minimal photobleaching and phototoxicity, allowing in vivo visualization of a variety of tissues and processes, ranging from developing hearts in live zebrafish embryos to ex vivo interrogation of the microarchitecture of optically cleared neonatal hearts. Here, we highlight multiple applications of LSFM and discuss several studies that have allowed better characterization of developmental and pathological processes in multiple models and tissues. These findings demonstrate the capacity of multiscale light-sheet imaging to uncover cardiovascular developmental and regenerative phenomena.

Hemodynamic shear force has been implicated as modulating Notch signaling-mediated cardiac trabeculation. Whether the spatiotemporal variations in wall shear stress (WSS) coordinate the initiation of trabeculation to influence ventricular contractile function remains unknown. Using light-sheet fluorescent microscopy, we reconstructed the 4D moving domain and applied computational fluid dynamics to quantify 4D WSS along the trabecular ridges and in the groves. In WT zebrafish, pulsatile shear stress developed along the trabecular ridges, with prominent endocardial Notch activity at 3 days after fertilization (dpf), and oscillatory shear stress developed in the trabecular grooves, with epicardial Notch activity at 4 dpf. Genetic manipulations were performed to reduce hematopoiesis and inhibit atrial contraction to lower WSS in synchrony with attenuation of oscillatory shear index (OSI) during ventricular development. γ-Secretase inhibitor of Notch intracellular domain (NICD) abrogated endocardial and epicardial Notch activity. Rescue with NICD mRNA restored Notch activity sequentially from the endocardium to trabecular grooves, which was corroborated by observed Notch-mediated cardiomyocyte proliferations on WT zebrafish trabeculae. We also demonstrated in vitro that a high OSI value correlated with upregulated endothelial Notch-related mRNA expression. In silico computation of energy dissipation further supports the role of trabeculation to preserve ventricular structure and contractile function. Thus, spatiotemporal variations in WSS coordinate trabecular organization for ventricular contractile function.

Purpose of Review: Real-time 3-dimensional (3-D) imaging of cardiovascular injury and regeneration remains challenging. We introduced a multi-scale imaging strategy that uses light-sheet illumination to enable applications of cardiovascular injury and repair in models ranging from zebrafish to rodent hearts. Recent Findings: Light-sheet imaging enables rapid data acquisition with high spatiotemporal resolution and with minimal photo-bleaching or photo-toxicity. We demonstrated the capacity of this novel light-sheet approach for scanning a region of interest with specific fluorescence contrast, thereby providing axial and temporal resolution at the cellular level without stitching image columns or pivoting illumination beams during one-time imaging. This cutting-edge imaging technique allows for elucidating the differentiation of stem cells in cardiac regeneration, providing an entry point to discover novel micro-circulation phenomenon with clinical significance for injury and repair. Summary: These findings demonstrate the multi-scale applications of this novel light-sheet imaging strategy to advance research in cardiovascular development and regeneration.

Pacemakers have existed for decades as a means to restore cardiac electrical rhythms. However, lead-related complications have remained a clinical challenge. While market-released leadless devices have addressed some of the issues, their pacer-integrated batteries cause new health risks and functional limitations. Inductive power transfer enables wireless powering of bioelectronic devices; however, Specific Absorption Rate and size limitations reduce power efficiency for biomedical applications. We designed a remote-controlled system in which power requirements were significantly reduced via intermittent power transfer to control stimulation intervals. In parallel, the cardiac component was miniaturized to facilitate intravascular deployment into the anterior cardiac vein. Given size constraints, efficiency was optimal via a circular receiver coil wrapped into a half-cylinder with a meandering tail. The pacemaker was epicardially tested in a euthanized pig at 60 beats per minute, 2 V amplitude, and 1 ms pulse width, restoring mean arterial pressure from 0 to 37 mmHg. Power consumption was 1 mW at a range of > 3 cm with no misalignment and at 2 cm with 45° displacement misalignment, 45° x-axis angular misalignment, or 45° y-axis angular misalignment. Thus, we demonstrated a remote-controlled miniaturized pacing system with low power consumption, thereby providing a basis for the next generation of wireless implantable devices.

Four-point electrode systems are commonly used for electric impedance measurements of biomaterials and tissues. We introduce a 2-point system to reduce electrode polarization for heterogeneous measurements of vascular wall. Presence of endoluminal oxidized low density lipoprotein (oxLDL) and lipids alters the electrochemical impedance that can be measured by electrochemical impedance spectroscopy (EIS). We developed a catheter-based 2-point micro-electrode configuration for intravascular deployment in New Zealand White rabbits. An array of 2 flexible round electrodes, 240µm in diameter and separated by 400µm was microfabricated and mounted on an inflatable balloon catheter for EIS measurement of the oxLDL-rich lesions developed as a result of high-fat diet-induced hyperlipidemia. Upon balloon inflation, the 2-point electrode array conformed to the arterial wall to allow deep intraplaque penetration via alternating current (AC). The frequency sweep from 10 to 300kHz generated an increase in capacitance, providing distinct changes in both impedance (Ω) and phase (ϕ) in relation to varying degrees of intraplaque lipid burden in the aorta. Aortic endoluminal EIS measurements were compared with epicardial fat tissue and validated by intravascular ultrasound and immunohistochemistry for plaque lipids and foam cells. Thus, we demonstrate a new approach to quantify endoluminal EIS via a 2-point stretchable electrode strategy.

Light Sheet Fluorescence Microscopy (LSFM) enables multi-dimensional and multi-scale imaging via illuminating specimens with a separate thin sheet of laser. It allows rapid plane illumination for reduced photo-damage and superior axial resolution and contrast. We hereby demonstrate cardiac LSFM (c-LSFM) imaging to assess the functional architecture of zebrafish embryos with a retrospective cardiac synchronization algorithm for four-dimensional reconstruction (3-D space + time). By combining our approach with tissue clearing techniques, we reveal the entire cardiac structures and hypertrabeculation of adult zebrafish hearts in response to doxorubicin treatment. By integrating the resolution enhancement technique with c-LSFM to increase the resolving power under a large field-of-view, we demonstrate the use of low power objective to resolve the entire architecture of large-scale neonatal mouse hearts, revealing the helical orientation of individual myocardial fibers. Therefore, our c-LSFM imaging approach provides multi-scale visualization of architecture and function to drive cardiovascular research with translational implication in congenital heart diseases.

Purpose of review: Blood flow is intimately linked with cardiovascular development, repair and dysfunction. The current review will build on the fluid mechanical principle underlying haemodynamic shear forces, mechanotransduction and metabolic effects. Recent findings: Pulsatile flow produces both time (∂τ/∂t) and spatial-varying shear stress (∂τ/∂x) to modulate vascular oxidative stress and inflammatory response with pathophysiological significance to atherosclerosis. The characteristics of haemodynamic shear forces, namely, steady laminar (∂τ/∂t = 0), pulsatile shear stress (PSS: unidirectional forward flow) and oscillatory shear stress (bidirectional with a near net 0 forward flow), modulate mechano-signal transduction to influence metabolic effects on vascular endothelial function. Atheroprotective PSS promotes antioxidant, anti-inflammatory and antithrombotic responses, whereas atherogenic oscillatory shear stress induces nicotinamide adenine dinucleotide phosphate oxidase–JNK signalling to increase mitochondrial superoxide production, protein degradation of manganese superoxide dismutase and post-translational protein modifications of LDL particles in the disturbed flow-exposed regions of vasculature. In the era of tissue regeneration, shear stress has been implicated in reactivation of developmental genes, namely, Wnt and Notch signalling, for vascular development and repair. Summary: Blood flow imparts a dynamic continuum from vascular development to repair. Augmentation of PSS confers atheroprotection and reactivation of developmental signalling pathways for regeneration.

PET myocardial perfusion imaging (MPI) is increasingly being used for noninvasive detection and evaluation of coronary artery disease. However, the widespread use of PET MPI has been limited by the shortcomings of the current PET perfusion tracers. The availability of these tracers is limited by the need for an onsite ((15)O water and (13)N ammonia) or nearby ((13)N ammonia) cyclotron or commitment to costly generators ((82)Rb). Owing to the short half-lives, such as 76 seconds for (82)Rb, 2.06 minutes for (15)O water, and 9.96 minutes for (13)N ammonia, their use in conjunction with treadmill exercise stress testing is either not possible ((82)Rb and (15)O water) or not practical ((13)N ammonia). Furthermore, the long positron range of (82)Rb makes image resolution suboptimal and its low myocardial extraction limits its defect resolution. In recent years, development of an (18)F-labeled PET perfusion tracer has gathered considerable interest. The longer half-life of (18)F (109 minutes) would make the tracer available as a unit dose from regional cyclotrons and allow use in conjunction with treadmill exercise testing. Furthermore, the short positron range of (18)F would result in better image resolution. Flurpiridaz F 18 is by far the most thoroughly studied in animal models and is the only (18)F-based PET MPI radiotracer currently undergoing clinical evaluation. Preclinical and clinical experience with Flurpiridaz F 18 demonstrated a high myocardial extraction fraction, high image and defect resolution, high myocardial uptake, slow myocardial clearance, and high myocardial-to-background contrast that was stable over time-important properties of an ideal PET MPI radiotracer. Preclinical data from other (18)F-labeled myocardial perfusion tracers are encouraging.

Hemodynamic shear forces are intimately linked with cardiac development, during which trabeculae form a network of branching outgrowths from the myocardium. Mutations that alter Notch signaling also result in trabeculation defects. Here, we assessed whether shear stress modulates trabeculation to influence contractile function. Specifically, we acquired 4D (3D + time) images with light sheets by selective plane illumination microscopy (SPIM) for rapid scanning and deep axial penetration during zebrafish morphogenesis. Reduction of blood viscosity via gata1a morpholino oligonucleotides (MO) reduced shear stress, resulting in downregulation of Notch signaling and attenuation of trabeculation. Arrest of cardiomyocyte contraction either by troponin T type 2a (tnnt2a) MO or in weak atriumm58 (wea) mutants resulted in reduced shear stress and downregulation of Notch signaling and trabeculation. Integrating 4D SPIM imaging with synchronization algorithm demonstrated that coinjection of neuregulin1 mRNA with gata1 MO rescued trabeculation to restore contractile function in association with upregulation of Notch-related genes. Crossbreeding of Tg(flk:mCherry) fish, which allows visualization of the vascular system with the Tg(tp1:gfp) Notch reporter line, revealed that shear stress-mediated Notch activation localizes to the endocardium. Deleting endocardium via the clochesk4 mutants downregulated Notch signaling, resulting in nontrabeculated ventricle. Subjecting endothelial cells to pulsatile flow in the presence of the ADAM10 inhibitor corroborated shear stress-activated Notch signaling to modulate trabeculation.

Common forms of atherosclerosis involve multiple genetic and environmental factors. While human genome-wide association studies have identified numerous loci contributing to coronary artery disease and its risk factors, these studies are unable to control environmental factors or examine detailed molecular traits in relevant tissues. We now report a study of natural variations contributing to atherosclerosis and related traits in over 100 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP). The mice were made hyperlipidemic by transgenic expression of human apolipoprotein E-Leiden (APOE-Leiden) and human cholesteryl ester transfer protein (CETP). The mice were examined for lesion size and morphology as well as plasma lipid, insulin and glucose levels, and blood cell profiles. A subset of mice was studied for plasma levels of metabolites and cytokines. We also measured global transcript levels in aorta and liver. Finally, the uptake of acetylated LDL by macrophages from HMDP mice was quantitatively examined. Loci contributing to the traits were mapped using association analysis, and relationships among traits were examined using correlation and statistical modeling. A number of conclusions emerged. First, relationships among atherosclerosis and the risk factors in mice resemble those found in humans. Second, a number of trait-loci were identified, including some overlapping with previous human and mouse studies. Third, gene expression data enabled enrichment analysis of pathways contributing to atherosclerosis and prioritization of candidate genes at associated loci in both mice and humans. Fourth, the data provided a number of mechanistic inferences; for example, we detected no association between macrophage uptake of acetylated LDL and atherosclerosis. Fifth, broad sense heritability for atherosclerosis was much larger than narrow sense heritability, indicating an important role for gene-by-gene interactions. Sixth, stepwise linear regression showed that the combined variations in plasma metabolites, including LDL/VLDL-cholesterol, trimethylamine N-oxide (TMAO), arginine, glucose and insulin, account for approximately 30 to 40% of the variation in atherosclerotic lesion area. Overall, our data provide a rich resource for studies of complex interactions underlying atherosclerosis.

CD4(+)CD25(+) regulatory T cells (T regs) play a major role in the maintenance of self-tolerance and immune suppression, although the mechanisms controlling T reg development and suppressor function remain incompletely understood. Herein, we provide evidence that Kruppel-like factor 10 (KLF10/TIEG1) constitutes an important regulator of T regulatory cell suppressor function and CD4(+)CD25(-) T cell activation through distinct mechanisms involving transforming growth factor (TGF)-beta1 and Foxp3. KLF10 overexpressing CD4(+)CD25(-) T cells induced both TGF-beta1 and Foxp3 expression, an effect associated with reduced T-Bet (Th1 marker) and Gata3 (Th2 marker) mRNA expression. Consistently, KLF10(-/-) CD4(+)CD25(-) T cells have enhanced differentiation along both Th1 and Th2 pathways and elaborate higher levels of Th1 and Th2 cytokines. Furthermore, KLF10(-/-) CD4(+)CD25(-) T cell effectors cannot be appropriately suppressed by wild-type T regs. Surprisingly, KLF10(-/-) T reg cells have reduced suppressor function, independent of Foxp3 expression, with decreased expression and elaboration of TGF-beta1, an effect completely rescued by exogenous treatment with TGF-beta1. Mechanistic studies demonstrate that in response to TGF-beta1, KLF10 can transactivate both TGF-beta1 and Foxp3 promoters, implicating KLF10 in a positive feedback loop that may promote cell-intrinsic control of T cell activation. Finally, KLF10(-/-) CD4(+)CD25(-) T cells promoted atherosclerosis by approximately 2-fold in ApoE(-/-)/scid/scid mice with increased leukocyte accumulation and peripheral pro-inflammatory cytokines. Thus, KLF10 is a critical regulator in the transcriptional network controlling TGF-beta1 in both CD4(+)CD25(-) T cells and T regs and plays an important role in regulating atherosclerotic lesion formation in mice.

Recent reports suggest dyslipidemia impairs dendritic cell (DC) function and adaptive immunity. This study aimed to characterize the effect of hypercholesterolemia on antigen-presenting cell function of DCs and DC-dependent CD4 + T-cell responses. DCs incubated in vitro with acetylated low-density lipoprotein cholesterol with or without an acyl-coenzyme A:cholesterol acyl-transferase inhibitor maintained their ability to prime CD4 + T cells. Analysis of T-cell proliferation and interferon-γ and tumor necrosis factor-α production after ex vivo coculture of naïve CD4 + T cells with splenic, inguinal, or iliac DCs from low-density lipoprotein receptor–deficient ( LDLR −/− ) or apolipoprotein E–deficient ( ApoE −/− ) mice fed an atherogenic diet highlighted DC efficacy in effector T-cell generation under hypercholesterolemic conditions. Adoptive transfer of carboxyfluorescein diacetate, succinimidyl ester (CFSE)-labeled naïve CD4 + T cells in LDLR −/− recipients and subsequent immunization demonstrated effective priming of naïve T cells in hypercholesterolemic mice. CFSE dilution analyses revealed that hypercholesterolemic DCs were equipotent in naïve CD4 + T-cell priming efficacy with normocholesterolemic DCs. Quantitative real-time PCR and flow cytometric analyses demonstrated that DC expression of multiple molecules involved in antigen processing, presentation, and T-cell stimulation remained unaltered by dyslipidemia. Finally, endogenous antigen-primed CD4 + T cells responded equivalently to a secondary ex vivo antigenic challenge, regardless of whether they were primed in vivo under hypercholesterolemic or control conditions, demonstrating that all essential steps in CD4 + T-cell responses remain intact under atherogenic conditions. This study affirms that the adaptive immune response prevails under the hypercholesterolemic conditions present in atherosclerosis. In particular, DCs remain functional antigen-presenting cells and maintain their ability to prime CD4 + T cells even when cholesterol-loaded.

Common forms of atherosclerosis involve multiple genetic and environmental factors. While human genome-wide association studies have identified numerous loci contributing to coronary artery disease and its risk factors, these studies are unable to control environmental factors or examine detailed molecular traits in relevant tissues. We now report a study of natural variations contributing to atherosclerosis and related traits in over 100 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP). The mice were made hyperlipidemic by transgenic expression of human apolipoprotein E-Leiden (APOE-Leiden) and human cholesteryl ester transfer protein (CETP). The mice were examined for lesion size and morphology as well as plasma lipid, insulin and glucose levels, and blood cell profiles. A subset of mice was studied for plasma levels of metabolites and cytokines. We also measured global transcript levels in aorta and liver. Finally, the uptake of acetylated LDL by macrophages from HMDP mice was quantitatively examined. Loci contributing to the traits were mapped using association analysis, and relationships among traits were examined using correlation and statistical modeling. A number of conclusions emerged. First, relationships among atherosclerosis and the risk factors in mice resemble those found in humans. Second, a number of trait-loci were identified, including some overlapping with previous human and mouse studies. Third, gene expression data enabled enrichment analysis of pathways contributing to atherosclerosis and prioritization of candidate genes at associated loci in both mice and humans. Fourth, the data provided a number of mechanistic inferences; for example, we detected no association between macrophage uptake of acetylated LDL and atherosclerosis. Fifth, broad sense heritability for atherosclerosis was much larger than narrow sense heritability, indicating an important role for gene-by-gene interactions. Sixth, stepwise linear regression showed that the combined variations in plasma metabolites, including LDL/VLDL-cholesterol, trimethylamine N-oxide (TMAO), arginine, glucose and insulin, account for approximately 30 to 40% of the variation in atherosclerotic lesion area. Overall, our data provide a rich resource for studies of complex interactions underlying atherosclerosis., Author Summary While recent genetic association studies in human populations have succeeded in identifying genetic loci that contribute to coronary artery disease (CAD) and related phenotypes, these loci explain only a small fraction of the genetic variation in CAD and associated traits. Here, we present a complementary approach using association analysis of atherosclerotic traits among inbred strains of mice. A strength of this approach is that it enables in-depth phenotypic characterization including gene expression and metabolic profiling across a variety of tissues, and integration of these molecular phenotypes with coronary artery disease itself. A striking finding was the large fraction of atherosclerosis that was explained by genetic interactions. Association analysis allowed us to identify genetic loci for atherosclerotic lesion area as well as transcript, cytokine and metabolite levels, and relationships among the traits were examined by correlation and network modeling. The plasma metabolites associated with atherosclerosis in mice, namely, LDL/VLDL-cholesterol, TMAO, arginine, glucose and insulin, overlapped with those observed in humans and accounted for approximately 30 to 40% of the observed variation in atherosclerotic lesion area. In summary, our data provide a detailed overview of the genetic architecture of atherosclerosis in mice and a rich resource for studies of the complex genetic and metabolic interactions that underlie the disease.