TAD evolutionary and functional characterization reveals diversity in mammalian TAD boundary properties and function.

Topological associating domains (TADs) are self-interacting genomic units crucial for shaping gene regulation patterns. Despite their importance, the extent of their evolutionary conservation and its functional implications remain largely unknown. In this study, we generate Hi-C and ChIP-seq data and compare TAD organization across four primate and four rodent species and characterize the genetic and epigenetic properties of TAD boundaries in correspondence to their evolutionary conservation. We find 14% of all human TAD boundaries to be shared among all eight species (ultraconserved), while 15% are human-specific. Ultraconserved TAD boundaries have stronger insulation strength, CTCF binding, and enrichment of older retrotransposons compared to species-specific boundaries. CRISPR-Cas9 knockouts of an ultraconserved boundary in a mouse model lead to tissue-specific gene expression changes and morphological phenotypes. Deletion of a human-specific boundary near the autism-related AUTS2 gene results in the upregulation of this gene in neurons. Overall, our study provides pertinent TAD boundary evolutionary conservation annotations and showcases the functional importance of TAD evolution.

Energetics of Cardiac Blood Flow in Hypertrophic Cardiomyopathy through Individualized Computational Modeling.

Hypertrophic cardiomyopathy (HCM) is a congenital heart disease characterized by thickening of the heart's left ventricle (LV) wall that can lead to cardiac dysfunction and heart failure. Ventricular wall thickening affects the motion of cardiac walls and blood flow within the heart. Because abnormal cardiac blood flow in turn could lead to detrimental remodeling of heart walls, aberrant ventricular flow patterns could exacerbate HCM progression. How blood flow patterns are affected by hypertrophy and inter-patient variability is not known. To address this gap in knowledge, we present here strategies to generate personalized computational fluid dynamics (CFD) models of the heart LV from patient cardiac magnetic resonance (cMR) images. We performed simulations of CFD LV models from three cases (one normal, two HCM). CFD computations solved for blood flow velocities, from which flow patterns and the energetics of flow within the LV were quantified. We found that, compared to a normal heart, HCM hearts exhibit anomalous flow patterns and a mismatch in the timing of energy transfer from the LV wall to blood flow, as well as changes in kinetic energy flow patterns. While our results are preliminary, our presented methodology holds promise for in-depth analysis of HCM patient hemodynamics in clinical practice.

Coaxial Placement of Balloon-Expandable and Self-Expanding Stents: Impact on Crush Resistance and Luminal Recovery in a Benchtop Model.

During endovascular interventions, coaxial deployment of stents may be required to preserve luminal gain. This study characterized in vitro the effect on crush resistance and postcompression recovery when 316L stainless steel balloon-expandable (BE) and laser-cut nitinol self-expanding (SE) venous stents were deployed coaxially. Various stent configurations were parallel-plate compressed from a fully expanded state to 50% diameter reduction (Criterion, Model 42; MTS, Eden Prairie, Minnesota) in a 37 °C ± 1 water bath. Coaxial deployments of SE stent inside BE stent and BE stent inside SE stent demonstrated higher crush resistances compared with each stent individually or their mathematical summation (analysis of variance P < .0001; pairwise comparison P < .01). The configuration of SE stent inside BE stent showed higher postcompression luminal recovery at 48.7% compared with that of BE stent inside SE stent at 27.5% (P = .0001). Coaxial deployment of SE stent inside BE stent may improve crush resistance and luminal recovery after compression in the appropriate clinical context.

TAD Evolutionary and functional characterization reveals diversity in mammalian TAD boundary properties and function.

Topological associating domains (TADs) are self-interacting genomic units crucial for shaping gene regulation patterns. Despite their importance, the extent of their evolutionary conservation and its functional implications remain largely unknown. In this study, we generate Hi-C and ChIP-seq data and compare TAD organization across four primate and four rodent species, and characterize the genetic and epigenetic properties of TAD boundaries in correspondence to their evolutionary conservation. We find that only 14% of all human TAD boundaries are shared among all eight species (ultraconserved), while 15% are human-specific. Ultraconserved TAD boundaries have stronger insulation strength, CTCF binding, and enrichment of older retrotransposons, compared to species-specific boundaries. CRISPR-Cas9 knockouts of two ultraconserved boundaries in mouse models leads to tissue-specific gene expression changes and morphological phenotypes. Deletion of a human-specific boundary near the autism-related AUTS2 gene results in upregulation of this gene in neurons. Overall, our study provides pertinent TAD boundary evolutionary conservation annotations, and showcase the functional importance of TAD evolution.

Effect of Blood Flow on Cardiac Morphogenesis and Formation of Congenital Heart Defects.

Congenital heart disease (CHD) affects about 1 in 100 newborns and its causes are multifactorial. In the embryo, blood flow within the heart and vasculature is essential for proper heart development, with abnormal blood flow leading to CHD. Here, we discuss how blood flow (hemodynamics) affects heart development from embryonic to fetal stages, and how abnormal blood flow solely can lead to CHD. We emphasize studies performed using avian models of heart development, because those models allow for hemodynamic interventions, in vivo imaging, and follow up, while they closely recapitulate heart defects observed in humans. We conclude with recommendations on investigations that must be performed to bridge the gaps in understanding how blood flow alone, or together with other factors, contributes to CHD.

Impact of Postthrombotic Vein Wall Biomechanics on Luminal Flow during Venous Angioplasty and Stent Placement: Computational Modeling Results.

This study characterized the impact of vein wall biomechanics on inflow diameter and luminal flow during venous angioplasty and stent placement, using postthrombotic and healthy biomechanical properties from an ovine venous stenosis and thrombosis model. Finite element analysis demonstrated more pronounced inflow channel narrowing in the postthrombotic vein compared with the healthy control vein during angioplasty and stent placement (relative inflow diameter reduction of 42% versus 13%, P < .0001). Computational fluid dynamics modeling showed increased relative areas of low wall shear rate in the postthrombotic vein compared with the normal vein (0.46 vs 0.24 for shear rate < 50 s-1; 0.13 vs 0.07 for shear rate < 15 s-1; P < .05), with flow stagnation and recirculation. Since inflow narrowing and low wall shear rate are associated with in-stent restenosis and reintervention, these computational results based on experimentally obtained biomechanical values highlight the significance of postthrombotic venous properties in optimizing venous intervention outcomes.

Dipeptidyl peptidase IV inhibition delays developmental programming of obesity and metabolic disease in male offspring of obese mothers.

Maternal obesity programs the offspring to metabolic diseases later in life; however, the mechanisms of programming are yet unclear, and no strategies exist for addressing its detrimental transgenerational effects. Obesity has been linked to dipeptidyl peptidase IV (DPPIV), an adipokine, and treatment of obese individuals with DPPIV inhibitors has been reported to prevent weight gain and improve metabolism. We hypothesized that DPPIV plays a role in maternal obesity-mediated programming. We measured plasma DPPIV activity in human maternal and cord blood samples from normal-weight and obese mothers at term. We found that maternal obesity increases maternal and cord blood plasma DPPIV activity but only in male offspring. Using two non-human primate models of maternal obesity, we confirmed the activation of DPPIV in the offspring of obese mothers. We then created a mouse model of maternal high-fat diet (HFD)-induced obesity, and found an early-life increase in plasma DPPIV activity in male offspring. Activation of DPPIV preceded the progression of obesity, glucose intolerance and insulin resistance in male offspring of HFD-fed mothers. We then administered sitagliptin, DPPIV inhibitor, to regular diet (RD)- and HFD-fed mothers, starting a week prior to breeding and continuing throughout pregnancy and lactation. We found that sitagliptin treatment of HFD-fed mothers delayed the progression of obesity and metabolic diseases in male offspring and had no effects on females. Our findings reveal that maternal obesity dysregulates plasma DPPIV activity in males and provide evidence that maternal inhibition of DPPIV has potential for addressing the transgenerational effects of maternal obesity.

Endovascular Venous Stenosis and Thrombosis Large Animal Model: Angiographic, Histological, and Biomechanical Characterizations.

PURPOSE: To characterize an ovine endovascular radiofrequency (RF) ablation-based venous stenosis and thrombosis model for studying venous biomechanics and response to intervention. MATERIALS AND METHODS: Unilateral short-segment (n = 2) or long-segment (n = 6) iliac vein stenoses were created in 8 adult sheep using an endovenous RF ablation technique. Angiographic assessment was performed at baseline, immediately after venous stenosis creation, and after 2-week (n = 6) or 3-month (n = 2) survival. Stenosed iliac veins and the contralateral healthy controls were harvested for histological and biomechanical assessment. RESULTS: At follow-up, the short-segment RF ablation group showed stable stenosis without occlusion. The long-segment group showed complete venous occlusion/thrombosis with the formation of collateral veins. Stenosed veins showed significant wall thickening (0.28 vs 0.16 mm, P = .0175) and confluent collagen deposition compared with the healthy controls. Subacute nonadherent thrombi were apparent at 2 weeks, which were replaced by fibrous luminal obliteration with channels of recanalization at 3 months. Stenosed veins demonstrated increased longitudinal stiffness (448.5 ± 5.4 vs 314.6 ± 1.5 kPa, P < .0001) and decreased circumferential stiffness (140.8 ± 2.6 vs 246.0 ± 1.6 kPa, P < .0001) compared with the healthy controls. CONCLUSION: Endovenous RF ablation is a reliable technique for creating venous stenosis and thrombosis in a large animal model with histological and biomechanical attributes similar to those seen in humans. This platform can facilitate understanding of venous biomechanics and testing of venous-specific devices and interventions.

Preclinical therapeutics ex ovo quail eggs as a biomimetic automation-ready xenograft platform.

Preclinical cancer research ranges from in vitro studies that are inexpensive and not necessarily reflective of the tumor microenvironment to mouse studies that are better models but prohibitively expensive at scale. Chorioallantoic membrane (CAM) assays utilizing Japanese quail (Coturnix japonica) are a cost-effective screening method to precede and minimize the scope of murine studies for anti-cancer efficacy and drug toxicity. To increase the throughput of CAM assays we have built and optimized an 11-day platform for processing up to 200 quail eggs per screening to evaluate drug efficacy and drug toxicity caused by a therapeutic. We demonstrate ex ovo concordance with murine in vivo studies, even when the in vitro and in vivo studies diverge, suggesting a role for this quail shell-free CAM xenograft assay in the validation of new anti-cancer agents.

Patient-specific computational modeling of endovascular aneurysm repair: State of the art and future directions.

Endovascular aortic repair (EVAR) has become the preferred intervention option for aortic aneurysms and dissections. This is because EVAR is much less invasive than the alternative open surgery repair. While in-hospital mortality rates are smaller for EVAR than open repair (1%-2% vs. 3%-5%), the early benefits of EVAR are lost after 3 years due to larger rates of complications in the EVAR group. Clinicians follow instructions for use (IFU) when possible, but are left with personal experience on how to best proceed and what choices to make with respect to stent-graft (SG) model choice, sizing, procedural options, and their implications on long-term outcomes. Computational modeling of SG deployment in EVAR and tissue remodeling after intervention offers an alternative way of testing SG designs in silico, in a personalized way before intervention, to ultimately select the strategies leading to better outcomes. Further, computational modeling can be used in the optimal design of SGs in cases of complex geometries. In this review, we address some of the difficulties and successes associated with computational modeling of EVAR procedures. There is still work to be done in all areas of EVAR in silico modeling, including model validation, before models can be applied in the clinic, but much progress has already been made. Critical to clinical implementation are current efforts focusing on developing fast algorithms that can achieve (near) real-time solutions, as well as ways of dealing with inherent uncertainties related to patient aortic wall degradation on an individualized basis. We are optimistic that EVAR modeling in the clinic will soon become a reality to help clinicians optimize EVAR interventions and ultimately reduce EVAR-associated complications.

Fetal Blood Flow and Genetic Mutations in Conotruncal Congenital Heart Disease.

In congenital heart disease, the presence of structural defects affects blood flow in the heart and circulation. However, because the fetal circulation bypasses the lungs, fetuses with cyanotic heart defects can survive in utero but need prompt intervention to survive after birth. Tetralogy of Fallot and persistent truncus arteriosus are two of the most significant conotruncal heart defects. In both defects, blood access to the lungs is restricted or non-existent, and babies with these critical conditions need intervention right after birth. While there are known genetic mutations that lead to these critical heart defects, early perturbations in blood flow can independently lead to critical heart defects. In this paper, we start by comparing the fetal circulation with the neonatal and adult circulation, and reviewing how altered fetal blood flow can be used as a diagnostic tool to plan interventions. We then look at known factors that lead to tetralogy of Fallot and persistent truncus arteriosus: namely early perturbations in blood flow and mutations within VEGF-related pathways. The interplay between physical and genetic factors means that any one alteration can cause significant disruptions during development and underscore our need to better understand the effects of both blood flow and flow-responsive genes.

Transient increase in VEGF-A leads to cardiac tube anomalies and increased risk of congenital heart malformations.

Vascular endothelial growth factor (VEGF) plays a critical role during early heart development. Clinical evidence shows that conditions associated with changes in VEGF signaling in utero are correlated with an increased risk of congenital heart defects (CHD) in newborns. However, how malformations develop after abnormal VEGF exposure is unknown. During embryogenesis, a primitive heart, consisting of an endocardial tube enveloped by a myocardial mantle, is the first organ to function. This tubular heart ultimately transforms into a four-chambered heart. To determine how a transient increase in VEGF prior to heart tube formation affects heart development leading to CHD, we applied exogenous VEGF or a control (vehicle) solution to quail embryos in ovo at Hamburger-Hamilton (HH) stage 8 (28-30 hr of incubation), right before heart tube formation. Light microscopy analysis of embryos re-incubated after treatment for 13 hrs (to approximately HH11/HH12) showed that increased VEGF leads to impaired heart tube elongation accompanied by diameter expansion. Micro-CT analysis of embryos re-incubated for 9 days (to approximately HH38), when the heart is fully formed, showed that VEGF treatment increased the rate of cardiac malformations in surviving embryos. Despite no sex differences in survival, female embryos were more likely to develop cardiac malformations. Our results further suggest that heart tube malformations after a transient increase in VEGF right before heart tube formation may be reversible, leading to normal hearts.

Multiscale cardiac imaging spanning the whole heart and its internal cellular architecture in a small animal model.

Cardiac pumping depends on the morphological structure of the heart, but also on its subcellular (ultrastructural) architecture, which enables cardiac contraction. In cases of congenital heart defects, localized ultrastructural disruptions that increase the risk of heart failure are only starting to be discovered. This is in part due to a lack of technologies that can image the three-dimensional (3D) heart structure, to assess malformations; and its ultrastructure, to assess organelle disruptions. We present here a multiscale, correlative imaging procedure that achieves high-resolution images of the whole heart, using 3D micro-computed tomography (micro-CT); and its ultrastructure, using 3D scanning electron microscopy (SEM). In a small animal model (chicken embryo), we achieved uniform fixation and staining of the whole heart, without losing ultrastructural preservation on the same sample, enabling correlative multiscale imaging. Our approach enables multiscale studies in models of congenital heart disease and beyond.

The heart is our hardest-working organ and beats around 100,000 times a day, pumping blood through a vast system of vessels to all areas of the body. Specialized heart cells make the heart contract rhythmically, enabling it to work efficiently. Contractile molecules inside these cells, called myofibrils, align within the heart cells, and heart cells align to each other, so that the heart tissue contracts effectively. However, when the heart has defects or is diseased this organization can be lost, and the heart may no longer pump blood efficiently, leading to sometimes life-threatening complications. For example, around one in a hundred newborn babies suffer from congenital heart defects, and despite medical advances, these conditions remain the main cause of non-infectious mortality in children. Many cases of congenital heart disease are diagnosed before a baby is born during an ultrasound scan. However, these scans, as well as subsequent diagnostic tools, lack the precision to detect problems within the heart cells. Now, Rykiel et al. used two complementary imaging techniques known as micro-computed tomography and scanning electron microscopy to acquire pictures of the whole heart as well as of the organization inside the heart cells. This made it possible to capture the structure of the heart tissue at both micrometer (the whole heart) and nanometer resolution (the inside of the cells), and to study what happens within the heart and its cells when the heart has a defect. Rykiel et al. tested the imaging technology on the hearts of chicken embryos, at stages equivalent to a five to six-month-old human fetus, and compared a healthy heart with a heart with a defect called tetralogy of Fallot. They found that the tissues in the heart with a defect had a sponge-like appearance, with increased space in between cells. Moreover, the myofibrils of the heart with a defect were aligned differently compared to those in the normal heart. More research is needed to fully understand what happens when the heart has a defect. However, the imaging technology used in this study offers the possibility of examining the heart at an unprecedented level of detail. This will deepen our understanding of how structural heart defects arise and how they affect the pumping of the heart, and will give us clues to design better treatments for patients with heart defects and other heart anomalies.

Predictors of Abdominal Aortic Aneurysm Risks.

Computational biomechanics via finite element analysis (FEA) has long promised a means of assessing patient-specific abdominal aortic aneurysm (AAA) rupture risk with greater efficacy than current clinically used size-based criteria. The pursuit stems from the notion that AAA rupture occurs when wall stress exceeds wall strength. Quantification of peak (maximum) wall stress (PWS) has been at the cornerstone of this research, with numerous studies having demonstrated that PWS better differentiates ruptured AAAs from non-ruptured AAAs. In contrast to wall stress models, which have become progressively more sophisticated, there has been relatively little progress in estimating patient-specific wall strength. This is because wall strength cannot be inferred non-invasively, and measurements from excised patient tissues show a large spectrum of wall strength values. In this review, we highlight studies that investigated the relationship between biomechanics and AAA rupture risk. We conclude that combining wall stress and wall strength approximations should provide better estimations of AAA rupture risk. However, before personalized biomechanical AAA risk assessment can become a reality, better methods for estimating patient-specific wall properties or surrogate markers of aortic wall degradation are needed. Artificial intelligence methods can be key in stratifying patients, leading to personalized AAA risk assessment.

Venous Biomechanics of Angioplasty and Stent Placement: Implications of the Poisson Effect.

PURPOSE: To characterize the Poisson effect in response to angioplasty and stent placement in veins and identify potential implications for guiding future venous-specific device design. MATERIALS AND METHODS: In vivo angioplasty and stent placement were performed in 3 adult swine by using an established venous stenosis model. Iron particle endothelium labeling was performed for real-time fluoroscopic tracking of the vessel wall during intervention. A finite-element computational model of a vessel was created with ADINA software (version 9.5) with arterial and venous biomechanical properties obtained from the literature to compare the response to radial expansion. RESULTS: In vivo angioplasty and stent placement in a venous stenosis animal model with iron particle endothelium labeling demonstrated longitudinal foreshortening that correlated with distance from the center of the balloon (R2 = 0.87) as well as adjacent segment narrowing that correlated with the increase in diameter of the treated stenotic segment (R2 = 0.89). Finite-element computational analysis demonstrated increased Poisson effect in veins relative to arteries (linear regression coefficient slope comparison, arterial slope 0.033, R2 = 0.9789; venous slope 0.204, R2 = 0.9975; P < .0001) as a result of greater longitudinal Young modulus in veins compared with arteries. CONCLUSIONS: Clinically observed adjacent segment narrowing during venous angioplasty and stent placement is a result of the Poisson effect, with redistribution of radially applied force to the longitudinal direction. The Poisson effect is increased in veins relative to arteries as a result of unique venous biomechanical properties, which may be relevant to consider in the design of future venous interventional devices.

Follow Me! A Tale of Avian Heart Development with Comparisons to Mammal Heart Development.

Avian embryos have been used for centuries to study development due to the ease of access. Because the embryos are sheltered inside the eggshell, a small window in the shell is ideal for visualizing the embryos and performing different interventions. The window can then be covered, and the embryo returned to the incubator for the desired amount of time, and observed during further development. Up to about 4 days of chicken development (out of 21 days of incubation), when the egg is opened the embryo is on top of the yolk, and its heart is on top of its body. This allows easy imaging of heart formation and heart development using non-invasive techniques, including regular optical microscopy. After day 4, the embryo starts sinking into the yolk, but still imaging technologies, such as ultrasound, can tomographically image the embryo and its heart in vivo. Importantly, because like the human heart the avian heart develops into a four-chambered heart with valves, heart malformations and pathologies that human babies suffer can be replicated in avian embryos, allowing a unique developmental window into human congenital heart disease. Here, we review avian heart formation and provide comparisons to the mammalian heart.

Optical coherence tomography for in vivo imaging of endocardial to mesenchymal transition during avian heart development.

The endocardial to mesenchymal transition (EndMT) that occurs in endocardial cushions during heart development is critical for proper heart septation and formation of the heart's valves. In EndMT, cells delaminate from the endocardium and migrate into the previously acellular endocardial cushions. Optical coherence tomography (OCT) imaging uses the optical properties of tissues for contrast, and during early development OCT can differentiate cellular versus acellular tissues. Here we show that OCT can be used to non-invasively track EndMT progression in vivo in the outflow tract cushions of chicken embryos. This enables in vivo studies to elucidate factors leading to cardiac malformations.

4-D Computational Modeling of Cardiac Outflow Tract Hemodynamics over Looping Developmental Stages in Chicken Embryos.

Cardiogenesis is interdependent with blood flow within the embryonic system. Recently, a number of studies have begun to elucidate the effects of hemodynamic forces acting upon and within cells as the cardiovascular system begins to develop. Changes in flow are picked up by mechanosensors in endocardial cells exposed to wall shear stress (the tangential force exerted by blood flow) and by myocardial and mesenchymal cells exposed to cyclic strain (deformation). Mechanosensors stimulate a variety of mechanotransduction pathways which elicit functional cellular responses in order to coordinate the structural development of the heart and cardiovascular system. The looping stages of heart development are critical to normal cardiac morphogenesis and have previously been shown to be extremely sensitive to experimental perturbations in flow, with transient exposure to altered flow dynamics causing severe late stage cardiac defects in animal models. This paper seeks to expand on past research and to begin establishing a detailed baseline for normal hemodynamic conditions in the chick outflow tract during these critical looping stages. Specifically, we will use 4-D (3-D over time) optical coherence tomography to create in vivo geometries for computational fluid dynamics simulations of the cardiac cycle, enabling us to study in great detail 4-D velocity patterns and heterogeneous wall shear stress distributions on the outflow tract endocardium. This information will be useful in determining the normal variation of hemodynamic patterns as well as in mapping hemodynamics to developmental processes such as morphological changes and signaling events during and after the looping stages examined here.

Few-shot hypercolumn-based mitochondria segmentation in cardiac and outer hair cells in focused ion beam-scanning electron microscopy (FIB-SEM) data.

We present a novel AI-based approach to the few-shot automated segmentation of mitochondria in large-scale electron microscopy images. Our framework leverages convolutional features from a pre-trained deep multilayer convolutional neural network, such as VGG-16. We then train a binary gradient boosting classifier on the resulting high-dimensional feature hypercolumns. We extract VGG-16 features from the first four convolutional blocks and apply bilinear upsampling to resize the obtained maps to the input image size. This procedure yields a 2688-dimensional feature hypercolumn for each pixel in a 224 × 224 input image. We then apply L 1-regularized logistic regression for supervised active feature selection to reduce dependencies among the features, to reduce overfitting, as well as to speed-up gradient boosting-based training. During inference we block process 1728 × 2022 large microscopy images. Our experiments show that in such a formulation of transfer learning our processing pipeline is able to achieve high-accuracy results on very challenging datasets containing a large number of irregularly shaped mitochondria in cardiac and outer hair cells. Our proposed few-shot training approach gives competitive performance with the state-of-the-art using far less training data.

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart.

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.