William J. Kowalski

Critical Transitions in Early Embryonic Aortic Arch Patterning and Hemodynamics

Transformation from the bilaterally symmetric embryonic aortic arches to the mature great vessels is a complex morphogenetic process, requiring both vasculogenic and angiogenic mechanisms. Early aortic arch development occurs simultaneously with rapid changes in pulsatile blood flow, ventricular function, and downstream impedance in both invertebrate and vertebrate species. These dynamic biomechanical environmental landscapes provide critical epigenetic cues for vascular growth and remodeling. In our previous work, we examined hemodynamic loading and aortic arch growth in the chick embryo at Hamburger-Hamilton stages 18 and 24. We provided the first quantitative correlation between wall shear stress (WSS) and aortic arch diameter in the developing embryo, and observed that these two stages contained different aortic arch patterns with no inter-embryo variation. In the present study, we investigate these biomechanical events in the intermediate stage 21 to determine insights into this critical transition. We performed fluorescent dye microinjections to identify aortic arch patterns and measured diameters using both injection recordings and high-resolution optical coherence tomography. Flow and WSS were quantified with 3D computational fluid dynamics (CFD). Dye injections revealed that the transition in aortic arch pattern is not a uniform process and multiple configurations were documented at stage 21. CFD analysis showed that WSS is substantially elevated compared to both the previous (stage 18) and subsequent (stage 24) developmental time-points. These results demonstrate that acute increases in WSS are followed by a period of vascular remodeling to restore normative hemodynamic loading. Fluctuations in blood flow are one possible mechanism that impacts the timing of events such as aortic arch regression and generation, leading to the variable configurations at stage 21. Aortic arch variations noted during normal rapid vascular remodeling at stage 21 identify a temporal window of increased vulnerability to aberrant aortic arch morphogenesis with the potential for profound effects on subsequent cardiovascular morphogenesis.

Analysis of early embryonic great-vessel microcirculation in zebrafish using high-speed confocal μPIV.

In the developing cardiovascular system, hemodynamic vascular loading is critical for angiogenesis and cardiovascular adaptation. Normal zebrafish embryos with transgenically-labeled endothelial and red blood cells provide an excellent in vivo model for studying the fluid-flow induced vascular loading. To characterize the developmental hemodynamics of early embryonic great-vessel microcirculation in the zebrafish embryo, two complementary studies (experimental and numerical) are presented. Quantitative comparison of the wall shear stress (WSS) at the first aortic arch (AA1) of wild-type zebrafish embryos during two consecutive developmental stages is presented, using time-resolved confocal micro-particle image velocimetry (μPIV). Analysis showed that there was significant WSS difference between 32 and 48 h post-fertilization (hpf) wild-type embryos, which correlates with normal arch morphogenesis. The vascular distensibility of the arch wall at systole and the acceleration/deceleration rates of time-lapse phase-averaged streamwise blood flow curves were also analyzed. To estimate the influence of a novel intermittent red-blood cell (RBC) loading on the endothelium, a numerical two-phase, volume of fluid (VOF) flow model was further developed with realistic in vivo conditions. These studies showed that near-wall effects and cell clustering increased WSS augmentation at a minimum of 15% when the distance of RBC from arch vessel wall was less than 3 μm or when RBC cell-to-cell distance was less than 3 μm. When compared to a smooth wall, the WSS augmentation increased by a factor of ~1.4 due to the roughness of the wall created by the endothelial cell profile. These results quantitatively highlight the contribution of individual RBC flow patterns on endothelial WSS in great-vessel microcirculation and will benefit the quantitative understanding of mechanotransduction in embryonic great vessel biology, including arteriovenous malformations (AVM).

Investigating developmental cardiovascular biomechanics and the origins of congenital heart defects.

Innovative research on the interactions between biomechanical load and cardiovascular (CV) morphogenesis by multiple investigators over the past 3 decades, including the application of bioengineering approaches, has shown that the embryonic heart adapts both structure and function in order to maintain cardiac output to the rapidly growing embryo. Acute adaptive hemodynamic mechanisms in the embryo include the redistribution of blood flow within the heart, dynamic adjustments in heart rate and developed pressure, and beat to beat variations in blood flow and vascular resistance. These biomechanically relevant events occur coincident with adaptive changes in gene expression and trigger adaptive mechanisms that include alterations in myocardial cell growth and death, regional and global changes in myocardial architecture, and alterations in central vascular morphogenesis and remodeling. These adaptive mechanisms allow the embryo to survive these biomechanical stresses (environmental, maternal) and to compensate for developmental errors (genetic). Recent work from numerous laboratories shows that a subset of these adaptive mechanisms is present in every developing multicellular organism with a “heart” equivalent structure. This chapter will provide the reader with an overview of some of the approaches used to quantify embryonic CV functional maturation and performance, provide several illustrations of experimental interventions that explore the role of biomechanics in the regulation of CV morphogenesis including the role of computational modeling, and identify several critical areas for future investigation as available experimental models and methods expand.

The myocardial regenerative potential of three-dimensional engineered cardiac tissues composed of multiple human iPS cell-derived cardiovascular cell lineages

Human induced pluripotent stem cells (hiPSCs) are a robust source for cardiac regenerative therapy due to their potential to support autologous and allogeneic transplant paradigms. The in vitro generation of three-dimensional myocardial tissue constructs using biomaterials as an implantable hiPSC-derived myocardium provides a path to realize sustainable myocardial regeneration. We generated engineered cardiac tissues (ECTs) from three cellular compositions of cardiomyocytes (CMs), endothelial cells (ECs), and vascular mural cells (MCs) differentiated from hiPSCs. We then determined the impact of cell composition on ECT structural and functional properties. In vitro force measurement showed that CM+EC+MC ECTs possessed preferential electromechanical properties versus ECTs without vascular cells indicating that incorporation of vascular cells augmented tissue maturation and function. The inclusion of MCs facilitated more mature CM sarcomeric structure, preferential alignment, and activated multiple tissue maturation pathways. The CM+EC+MC ECTs implanted onto infarcted, immune tolerant rat hearts engrafted, displayed both host and graft-derived vasculature, and ameliorated myocardial dysfunction. Thus, a composition of CMs and multiple vascular lineages derived from hiPSCs and incorporated into ECTs promotes functional maturation and demonstrates myocardial replacement and perfusion relevant for clinical translation.

Transition from fetal to neonatal circulation: Modeling the effect of umbilical cord clamping.

Hemodynamics of the fetal to neonatal transition are orchestrated through complex physiological changes and results in cardiovascular adaptation to the adult biventricular circulation. Clinical practice during this critical period can influence vital organ physiology for normal newborns, premature babies and congenital heart defect patients. Particularly, the timing of the cord clamping procedure, immediate (ICC) vs. delayed cord clamping (DCC), is hypothesized to be an important factor for the transitory fetal hemodynamics. The clinical need for a quantitative understanding of this physiology motivated the development of a lumped parameter model (LPM) of the fetal cardio-respiratory system covering the late-gestation to neonatal period. The LPM was validated with in vivo clinical data and then used to predict the effects of cord clamping procedures on hemodynamics and vital gases. Clinical time-dependent resistance functions to simulate the vascular changes were introduced. For DCC, placental transfusion (31.3 ml) increased neonatal blood volume by 11.7%. This increased blood volume is reflected in an increase in preload pressures by ~20% compared to ICC, which in turn increased the cardiac output (CO) by 20% (COICC=993 ml/min; CODCC=1197 ml/min). Our model accurately predicted dynamic flow patterns in vivo. DCC was shown to maintain oxygenation if the onset of pulmonary respiration was delayed or impaired. On the other hand, a significant 25% decrease in oxygen saturations was observed when applying ICC under the same physiological conditions. We conclude that DCC has a significant impact on newborn hemodynamics, mainly because of the improved blood volume and the sustained placental respiration.

Left atrial ligation alters intracardiac flow patterns and the biomechanical landscape in the chick embryo.

Background: Hypoplastic left heart syndrome (HLHS) is a major human congenital heart defect that results in single ventricle physiology and high mortality. Clinical data indicate that intracardiac blood flow patterns during cardiac morphogenesis are a significant etiology. We used the left atrial ligation (LAL) model in the chick embryo to test the hypothesis that LAL immediately alters intracardiac flow streams and the biomechanical environment, preceding morphologic and structural defects observed in HLHS. Results: Using fluorescent dye injections, we found that intracardiac flow patterns from the right common cardinal vein, right vitelline vein, and left vitelline vein were altered immediately following LAL. Furthermore, we quantified a significant ventral shift of the right common cardinal and right vitelline vein flow streams. We developed an in silico model of LAL, which revealed that wall shear stress was reduced at the left atrioventricular canal and left side of the common ventricle. Conclusions: Our results demonstrate that intracardiac flow patterns change immediately following LAL, supporting the role of hemodynamics in the progression of HLHS. Sites of reduced WSS revealed by computational modeling are commonly affected in HLHS, suggesting that changes in the biomechanical environment may lead to abnormal growth and remodeling of left heart structures. Developmental Dynamics 243:652–662, 2014.

Computational hemodynamic optimization predicts dominant aortic arch selection is driven by embryonic outflow tract orientation in the chick embryo

In the early embryo, a series of symmetric, paired vessels, the aortic arches, surround the foregut and distribute cardiac output to the growing embryo and fetus. During embryonic development, the arch vessels undergo large-scale asymmetric morphogenesis to form species-specific adult great vessel patterns. These transformations occur within a dynamic biomechanical environment, which can play an important role in the development of normal arch configurations or the aberrant arch morphologies associated with congenital cardiac defects. Arrested migration and rotation of the embryonic outflow tract during late stages of cardiac looping has been shown to produce both outflow tract and several arch abnormalities. Here, we investigate how changes in flow distribution due to a perturbation in the angular orientation of the embryonic outflow tract impact the morphogenesis and growth of the aortic arches. Using a combination of in vivo arch morphometry with fluorescent dye injection and hemodynamics-driven bioengineering optimization-based vascular growth modeling, we demonstrate that outflow tract orientation significantly changes during development and that the associated changes in hemodynamic load can dramatically influence downstream aortic arch patterning. Optimization reveals that balancing energy expenditure with diffusive capacity leads to multiple arch vessel patterns as seen in the embryo, while minimizing energy alone led to the single arch configuration seen in the mature arch of aorta. Our model further shows the critical importance of the orientation of the outflow tract in dictating morphogenesis to the adult single arch and accurately predicts arch IV as the dominant mature arch of aorta. These results support the hypothesis that abnormal positioning of the outflow tract during early cardiac morphogenesis may lead to congenital defects of the great vessels due to altered hemodynamic loading.

Impact of Cell Composition and Geometry on Human Induced Pluripotent Stem Cells-Derived Engineered Cardiac Tissue

The current study describes a scalable, porous large-format engineered cardiac tissue (LF-ECT) composed of human induced pluripotent stem cells (hiPSCs) derived multiple lineage cardiac cells with varied 3D geometries and cell densities developed towards the goal of scale-up for large animal pre-clinical studies. We explored multiple 15 × 15 mm ECT geometries using molds with rectangular internal staggered posts (mesh, ME), without posts (plain sheet, PS), or long parallel posts (multiple linear bundles, ML) and a gel matrix containing hiPSC-derived cardiomyocytes, endothelial, and vascular mural cells matured in vitro for 14 days. ME-ECTs displayed the lowest dead cell ratio (p < 0.001) and matured into 0.5 mm diameter myofiber bundles with greater 3D cell alignment and higher active stress than PS-ECTs. Increased initial ECT cell number beyond 6 M per construct resulted in reduced cell survival and lower active stress. The 6M-ME-ECTs implanted onto 1 week post-infarct immune tolerant rat hearts engrafted, displayed evidence for host vascular coupling, and recovered myocardial structure and function with reduced scar area. We generated a larger (30 × 30 mm) ME-ECT to confirm scalability. Thus, large-format ECTs generated from hiPSC-derived cardiac cells may be feasible for large animal preclinical cardiac regeneration paradigms.

Simultaneous real-time quantification of blood flow and vascular growth in the chick embryo using optical coherence tomography

Experimental and clinical data indicate that hemodynamic forces within the embryo provide critical biomechanical cues for cardiovascular morphogenesis, growth, and remodeling and that perturbed flow is a major etiology of congenital heart disease. However, embryonic flow-growth relationships are largely qualitative and poorly defined. In this work, we provide a quantitative analysis of in vivo flow and growth trends in the chick embryo using optical coherence tomography (OCT) to acquire simultaneous velocity and structural data of the right vitelline artery continuously over a ten hour period beginning at stage 16 (hour 54). We obtained 3D vessel volumes (15 μm lateral, 4.3 μm axial resolutions, 6 μm slice spacing) at 60 minute intervals, taking a B-scan time series totaling one cardiac cycle at each slice. Embryos were maintained at a constant 37°C and 60% humidity during the entire acquisition period through an inhouse built chamber. The 3D vessel lumen geometries were reconstructed manually to assess growth. Blood flow velocity was computed from the central B-scan using red blood cell particle image velocimetry. The use of extended OCT imaging as a non-invasive method for continuous and simultaneous flow and structural data can enhance our understanding of the biomechanical regulation of critical events in morphogenesis. Data acquired will be useful to validate predictive finite-element 3D growth models.

Hemodynamic flow visualization of early embryonic great vessels using μPIV

Microparticle image velocimetry (μPIV) is an evolving quantitative methodology to closely and accurately monitor the cardiac flow dynamics and mechanotransduction during vascular morphogenesis. While PIV technique has a long history, contemporary developments in advanced microscopy have significantly expanded its power. This chapter includes three new methods for μPIV acquisition in selected embryonic structures achieved through advanced optical imaging: (1) high-speed confocal scanning of transgenic zebrafish embryos, where the transgenic erythrocytes act as the tracing particles; (2) microinjection of artificial seeding particles in chick embryos visualized with stereomicroscopy; and (3) real-time, time-resolved optical coherence tomography acquisition of vitelline vessel flow profiles in chick embryos, tracking the erythrocytes.