Sandra Rugonyi

Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI

The protease thrombin is required for normal hemostasis and pathologic thrombogenesis. Since the mechanism of coagulation factor XI (FXI)-dependent thrombus growth remains unclear, we investigated the contribution of FXI to thrombus formation in a primate thrombosis model. Pretreatment of baboons with a novel anti-human FXI monoclonal antibody (aXIMab; 2 mg/kg) inhibited plasma FXI by at least 99% for 10 days, and suppressed thrombin-antithrombin (TAT) complex and beta-thromboglobulin (betaTG) formation measured immediately downstream from thrombi forming within collagen-coated vascular grafts. FXI inhibition with aXIMab limited platelet and fibrin deposition in 4-mm diameter grafts without an apparent increase in D-dimer release from thrombi, and prevented the occlusion of 2-mm diameter grafts without affecting template bleeding times. In comparison, pretreatment with aspirin (32 mg/kg) prolonged bleeding times but failed to prevent graft occlusion, supporting the concept that FXI blockade may offer therapeutic advantages over other antithrombotic agents in terms of bleeding complications. In whole blood, aXIMab prevented fibrin formation in a collagen-coated flow chamber, independent of factor XII and factor VII. These data suggest that endogenous FXI contributes to arterial thrombus propagation through a striking amplification of thrombin generation at the thrombus luminal surface.

The biophysical function of pulmonary surfactant

Pulmonary surfactant lowers surface tension in the lungs. Physiological studies indicate two key aspects of this function: that the surfactant film forms rapidly; and that when compressed by the shrinking alveolar area during exhalation, the film reduces surface tension to very low values. These observations suggest that surfactant vesicles adsorb quickly, and that during compression, the adsorbed film resists the tendency to collapse from the interface to form a 3D bulk phase. Available evidence suggests that adsorption occurs by way of a rate-limiting structure that bridges the gap between the vesicle and the interface, and that the adsorbed film avoids collapse by undergoing a process of solidification. Current models, although incomplete, suggest mechanisms that would partially explain both rapid adsorption and resistance to collapse as well as how different constituents of pulmonary surfactant might affect its behavior.

Changes in wall motion and blood flow in the outflow tract of chick embryonic hearts observed with optical coherence tomography after outflow tract banding and vitelline-vein ligation

Optical coherence tomography (OCT) is a non-contact, non-invasive and high-resolution imaging technique, suited to study early cardiovascular development. Alterations in hemodynamic conditions during early development are known to lead to cardiac defects, presumably as a result of changes in cardiac biomechanics produced by the alterations. In this paper, we demonstrate the use of a spectral domain OCT in visualizing and quantifying changes in cardiac wall motion and blood-flow velocities under normal and altered hemodynamic conditions in chicken embryos at an early stage of development (Hamburger-Hamilton stage HH18, approximately 3 days of incubation), focusing on the heart outflow tract (OFT). The OCT system employed acquired simultaneously microstructural and blood-flow images at a rate of 92 frames s(-1)with a spatial resolution of approximately 10 microm. OCT imaging allowed in vivo visualization of the OFT microstructures, e.g. the lumen, cardiac cushions and myocardium. We found that alterations in hemodynamic conditions, through OFT banding and vitelline-vein ligation, changed blood-flow velocities through the OFT, as expected. Further, OCT allowed quantification of changes in the dynamics of OFT wall motion. Our results therefore establish the utility of spectral domain OCT to study the influence of hemodynamic conditions on heart development in intact, in vivo chicken embryo models.

Measurement of absolute blood flow velocity in outflow tract of HH18 chicken embryo based on 4D reconstruction using spectral domain optical coherence tomography.

The measurement of blood-plasma absolute velocity distributions with high spatial and temporal resolution in vivo is important for the investigation of embryonic heart at its early stage of development. We introduce a novel method to measure absolute blood flow velocity based on high speed spectral domain optical coherence tomography (OCT) and apply it to measure velocities across the heart outflow tract (OFT) of a chicken embryo (stage HH18). First, we use the OCT system to acquire 4D 
[(x,y,z) + t] images of the OFT in vivo. Second, we reconstruct the 4D microstructural images and obtain the orientation of the OFT at its maximum expansion, from which the centerline of the OFT is calculated based on the OFT boundary segmentation. Assuming flow is parallel to the vessel orientation, the obtained centerline indicates the flow direction. Finally, the absolute flow velocity is evaluated based on the direction given by the centerline and the axial velocity obtained from Doppler OCT. Using this method, we compare flow velocity profiles at various positions along the chicken embryo OFT.

Efficient postacquisition synchronization of 4-D nongated cardiac images obtained from optical coherence tomography: application to 4-D reconstruction of the chick embryonic heart.

Four-dimensional (4-D) imaging of the embryonic heart allows study of cardiac morphology and function in vivo during development. However, 4-D imaging of the embryonic heart using current techniques, including optical coherence tomography (OCT), is limited by the rate of image acquisition. Here, we present a nongated 4-D imaging strategy combined with an efficient postacquisition synchronization procedure that circumvents limitations on acquisition rate. The 4-D imaging strategy acquires a time series of images in B mode at several different locations along the heart, rendering out-of-phase image sequences. Then, our synchronization procedure uses similarity of local structures to find the phase shift between neighboring image sequences, employing M-mode images (extracted from the acquired B-mode images) to achieve computational efficiency. Furthermore, our procedure corrects the phase shifts by considering the phase lags introduced by peristaltic-like contractions of the embryonic heart wall. We applied the 4-D imaging strategy and synchronization procedure to reconstruct the cardiac outflow tract (OFT) of a chick embryo, imaged with OCT at early stages of development (Hamburger-Hamilton stage 18). We showed that the proposed synchronization procedure achieves efficiency without sacrificing accuracy and that the reconstructed 4-D images properly captured the dynamics of the OFT wall motion.

Biomechanics of early cardiac development

Biomechanics affect early cardiac development, from looping to the development of chambers and valves. Hemodynamic forces are essential for proper cardiac development, and their disruption leads to congenital heart defects. A wealth of information already exists on early cardiac adaptations to hemodynamic loading, and new technologies, including high-resolution imaging modalities and computational modeling, are enabling a more thorough understanding of relationships between hemodynamics and cardiac development. Imaging and modeling approaches, used in combination with biological data on cell behavior and adaptation, are paving the road for new discoveries on links between biomechanics and biology and their effect on cardiac development and fetal programming.

Biomechanics of the Chick Embryonic Heart Outflow Tract at HH18 Using 4D Optical Coherence Tomography Imaging and Computational Modeling

During developmental stages, biomechanical stimuli on cardiac cells modulate genetic programs, and deviations from normal stimuli can lead to cardiac defects. Therefore, it is important to characterize normal cardiac biomechanical stimuli during early developmental stages. Using the chicken embryo model of cardiac development, we focused on characterizing biomechanical stimuli on the Hamburger–Hamilton (HH) 18 chick cardiac outflow tract (OFT), the distal portion of the heart from which a large portion of defects observed in humans originate. To characterize biomechanical stimuli in the OFT, we used a combination of in vivo optical coherence tomography (OCT) imaging, physiological measurements and computational fluid dynamics (CFD) modeling. We found that, at HH18, the proximal portion of the OFT wall undergoes larger circumferential strains than its distal portion, while the distal portion of the OFT wall undergoes larger wall stresses. Maximal wall shear stresses were generally found on the surface of endocardial cushions, which are protrusions of extracellular matrix onto the OFT lumen that later during development give rise to cardiac septa and valves. The non-uniform spatial and temporal distributions of stresses and strains in the OFT walls provide biomechanical cues to cardiac cells that likely aid in the extensive differential growth and remodeling patterns observed during normal development.

Congenital heart malformations induced by hemodynamic altering surgical interventions.

Embryonic heart formation results from a dynamic interplay between genetic and environmental factors. Blood flow during early embryonic stages plays a critical role in heart development, as interactions between flow and cardiac tissues generate biomechanical forces that modulate cardiac growth and remodeling. Normal hemodynamic conditions are essential for proper cardiac development, while altered blood flow induced by surgical manipulations in animal models result in heart defects similar to those seen in humans with congenital heart disease. This review compares the altered hemodynamics, changes in tissue properties, and cardiac defects reported after common surgical interventions that alter hemodynamics in the early chick embryo, and shows that interventions produce a wide spectrum of cardiac defects. Vitelline vein ligation and left atrial ligation decrease blood pressure and flow; and outflow tract banding increases blood pressure and flow velocities. These three surgical interventions result in many of the same cardiac defects, which indicate that the altered hemodynamics interfere with common looping, septation and valve formation processes that occur after intervention and that shape the four-chambered heart. While many similar defects develop after the interventions, the varying degrees of hemodynamic load alteration among the three interventions also result in varying incidence and severity of cardiac defects, indicating that the hemodynamic modulation of cardiac developmental processes is strongly dependent on hemodynamic load.

Hyperglycemia Slows Embryonic Growth and Suppresses Cell Cycle via Cyclin D1 and p21

In pregnant women, the diabetic condition results in a three- to fivefold increased risk for fetal cardiac malformations as a result of elevated glucose concentrations and the resultant osmotic stress in the developing embryo and fetus. Heart development before septation in the chick embryo was studied under two hyperglycemic conditions. Pulsed hyperglycemia induced by daily administration of glucose during 3 days of development caused daily spikes in plasma glucose concentration. In a second model, sustained hyperglycemia was induced with a single injection of glucose into the yolk on day 0. The sustained model raised the average plasma glucose concentration from 70 mg/dL to 180 mg/dL and led to decreased gene expression of glucose transporter GLUT1. Both models of hyperglycemia reduced embryo size, increased mortality, and delayed development. Within the heart outflow tract, reduced proliferation of myocardial and endocardial cells resulted from the sustained hyperglycemia and hyperosmolarity. The cell cycle inhibitor p21 was significantly increased, whereas cyclin D1, a cell cycle promoter, decreased in sustained hyperglycemia compared with controls. The evidence suggests that hyperglycemia-induced developmental delays are associated with slowed cell cycle progression, leading to reduced cellular proliferation. The suppression of critical developmental steps may underlie the cardiac defects observed during late gestation under hyperglycemic conditions.

Assessment of strain and strain rate in embryonic chick heart in vivo using tissue Doppler optical coherence tomography

We present a method to assess the in vivo radial strain and strain rate of the myocardial wall, which is of great importance to understand the biomechanics of cardiac development, using tissue Doppler optical coherence tomography (tissue-DOCT). Combining the structure and velocity information acquired from tissue-DOCT, the velocity distribution in the myocardial wall is plotted, from which the radial strain and strain rate are evaluated. The results demonstrate that tissue-DOCT can be used as a useful tool to describe tissue deformation, especially, the biomechanical characteristics of the embryonic heart.