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The role of hemodynamic forces within the embryo as biomechanical regulators for cardiovascular morphogenesis, growth, and remodeling is well supported through the experimental studies. Furthermore, clinical experience suggests that perturbed flow disrupts the normal vascular growth process as one etiology for congenital heart diseases (CHD) and for fetal adaptation to CHD. However, the relationships between hemodynamics, gene expression and embryonic vascular growth are poorly defined due to the lack of concurrent, sequential in vivo data. In this study, a long-term, time-lapse optical coherence tomography (OCT) imaging campaign was conducted to acquire simultaneous blood velocity, pulsatile micro-pressure and morphometric data for 3 consecutive early embryonic stages in the chick embryo. In conjunction with the in vivo growth and hemodynamics data, in vitro reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to track changes in transcript expression relevant to histogenesis and remodeling of the embryonic arterial wall. Our non-invasive extended OCT imaging technique for the microstructural data showed continuous vessel growth. OCT data coupled with the PIV technique revealed significant but intermitted increases in wall shear stress (WSS) between first and second assigned stages and a noticeable decrease afterwards. Growth rate, however, did not vary significantly throughout the embryonic period. Among all the genes studied, only the MMP-2 and CASP-3 expression levels remained unchanged during the time course. Concurrent relationships were obtained among the transcriptional modulation of the genes, vascular growth and hemodynamics-related changes. Further studies are indicated to determine cause and effect relationships and reversibility between mechanical and molecular regulation of vasculogenesis., Competing Interests: The authors have declared that no competing interests exist.

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.

The relative contributions of inflammation and ischemia to the pathogenesis of periventricular leukomalacia (PVL) have not been elucidated. We hypothesized that fetal cardiovascular function and cerebral blood flow velocity (BFV) would be decreased in a rat model of chorioamnionitis. We also tested whether placental inflammation was related to proximity to the cervix in our model of chorioamnionitis [intracervical lipopolysaccharide (LPS) or vehicle (PBS) injection]. On embryonic d 15, Sprague-Dawley rats underwent baseline maternal and fetal echocardiography, followed by LPS or PBS injection, then serial echocardiographic evaluations until embryonic day (ED) 21. One hour after birth, pups had middle cerebral artery (MCA) BFV measured. Placentas of LPS-exposed pups exhibited uniform, higher inflammation grades (p < 0.001). All fetal BFVs increased with advancing GA (p < 0.001) whereas resistance index (RI) decreased (p < 0.001). There was no difference in fetal BFV between the groups other than a reduced gestation-related increase in descending aorta BFV in LPS-exposed rats (p < 0.05). Newborn pups exposed to LPS had lower heart rate (p = 0.006) and MCA BFV (p = 0.024) and higher RI (p = 0.003) and pulsatility index (PI; p = 0.004). In conclusion, intracervical LPS injection produces an inflammation independent of placental position, a blunted increase in gestation-related aortic BFV, and a decrease in MCA BFV in newborn pups.

Caffeine consumption during pregnancy is reported to increase the risk of in utero growth restriction and spontaneous abortion. In the present study, we tested the hypothesis that modest maternal caffeine exposure affects in utero developing embryonic cardiovascular (CV) function and growth without altering maternal hemodynamics. Caffeine (10 mg.kg(-1).day(-1) subcutaneous) was administered daily to pregnant CD-1 mice from embryonic days (EDs) 9.5 to 18.5 of a 21-day gestation. We assessed maternal and embryonic CV function at baseline and at peak maternal serum caffeine concentration using high-resolution echocardiography on EDs 9.5, 11.5, 13.5, and 18.5. Maternal caffeine exposure did not influence maternal body weight gain, maternal CV function, or embryo resorption. However, crown-rump length and body weight were reduced in maternal caffeine treated embryos by ED 18.5 (P < 0.05). At peak maternal serum caffeine concentration, embryonic carotid artery, dorsal aorta, and umbilical artery flows transiently decreased from baseline at ED 11.5 (P < 0.05). By ED 13.5, embryonic aortic and umbilical artery flows were insensitive to the peak maternal caffeine concentration; however, the carotid artery flow remained affected. By ED 18.5, baseline embryonic carotid artery flow increased and descending aortic flow decreased versus non-caffeine-exposed embryos. Maternal treatment with the adenosine A(2A) receptor inhibitor reproduced the embryonic hemodynamic effects of maternal caffeine exposure. Adenosine A(2A) receptor gene expression levels of ED 11.5 embryo and ED 18.5 uterus were decreased. Results suggest that modest maternal caffeine exposure has adverse effects on developing embryonic CV function and growth, possibly mediated via adenosine A(2A) receptor blockade.

Normal blood flow is essential for proper heart formation during embryonic development, as abnormal hemodynamic load (blood pressure and shear stress) results in cardiac defects seen in congenital heart disease. However, the progressive detrimental remodeling processes that relate altered blood flow to cardiac defects remain unclear. Endothelial-mesenchymal cell transition is one of the many complex developmental events involved in transforming the early embryonic outflow tract into the aorta, pulmonary trunk, interventricular septum, and semilunar valves. This study elucidated the effects of increased hemodynamic load on endothelial-mesenchymal transition remodeling of the outflow tract cushions in vivo. Outflow tract banding was used to increase hemodynamic load in the chicken embryo heart between Hamburger and Hamilton stages 18 and 24. Increased hemodynamic load induced increased cell density in outflow tract cushions, fewer cells along the endocardial lining, endocardium junction disruption, and altered periostin expression as measured by confocal microscopy analysis. In addition, 3D focused ion beam scanning electron microscopy analysis determined that a portion of endocardial cells adopted a migratory shape after outflow tract banding that is more irregular, elongated, and with extensive cellular projections compared to normal cells. Proteomic mass-spectrometry analysis quantified altered protein composition after banding that is consistent with a more active stage of endothelial-mesenchymal transition. Outflow tract banding enhances the endothelial-mesenchymal transition phenotype during formation of the outflow tract cushions, suggesting that endothelial-mesenchymal transition is a critical developmental process that when disturbed by altered blood flow gives rise to cardiac malformation and defects. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

We investigate cardiovascular (CV) developmental physiology and biomechanics in order to understand the dramatic acquisition of form and function during normal development and to identify the adaptive mechanisms that allow embryos to survive adverse genetic and epigenetic events. Cardiovascular patterning, morphogenesis, and growth occur via highly conserved genetic mechanisms. Structural and functional maturation of the embryonic heart is also conserved across a broad range of species with evidence for load dependence from onset of the heartbeat. The embryonic heart dynamically adapts to changes in biomechanical loading conditions and for reasons not yet clear, adapts better to increased than to decreased mechanical load. In mammals, maternal cardiovascular function dynamically impacts embryonic/fetal growth and hemodynamics and these interactions can now be studied longitudinally using high-resolution noninvasive techniques. Maternal exposure to hypoxia and to bioactive chemicals, such as caffeine, can rapidly impact embryonic/fetal cardiovascular function, growth, and outcome. Finally, tissue engineering approaches can be applied to investigate basic developmental aspects of the embryonic myocardium. We use isolated embryonic and fetal chick, mouse, or rat cardiac cells to generate 3D engineered early embryonic cardiac tissues (EEECT). EEECT retains the morphologic and proliferative features of embryonic myocardium, responds to increased mechanical load with myocyte hyperplasia, and may be an excellent future material for use in cardiac repair and regeneration. These insights into cardiovascular embryogenesis are relevant to identifying mechanisms for congenital cardiovascular malformations and for developing cell- and tissue-based strategies for myocardial repair. [ABSTRACT FROM AUTHOR]

Aim: We tested the hypothesis that murine embryonic cardiovascular (CV) function is vulnerable to transient changes in maternal transplacental oxygen support during the critical period of CV morphogenesis. Methods: We measured maternal heart rate (MHR), maternal blood pressure (MBP), and embryonic heart rate (EHR) during mechanical ventilatory support, then induced transient maternal hypoxia daily from gestation day (ED) 10.5 to ED16.5 in pregnant ICR mice. Hypoxia was induced by suspending mechanical ventilation for 30 s or by the replacement of inspired oxygen with nitrogen (75% or 100%) for 30 s while maintaining ventilation. Results: We noted a rapid onset of maternal hypotension in response to hypoxia that quickly recovered following reoxygenation. Following a brief lag time that was not gestation specific, EHR decreased in response to hypoxia. The magnitude of embryo bradycardia and the rate of EHR decline and recovery displayed gestation specific patterns. The magnitude of embryo bradycardia was similar from ED10.5 to ED13.5 and then increased with gestation. Before ED13.5, only 40% of embryos recovered to the baseline EHR following transient maternal hypoxia (vs 80% of embryos after ED 13.5). EHR following recovery exceeded baseline EHR after ED15.5. Nitrogen inhalation (75% or 100%) produced changes in maternal and embryonic hemodynamics similar to suspended ventilation induced hypoxia. Conclusions: The mammalian embryo is vulnerable to transient decreases in maternal oxygenation during the critical period of organogenesis and the gestational specific EHR response to hypoxia may reflect both increased embryonic oxygen demand and the maturation of neurohumoral heart rate regulation. [ABSTRACT FROM AUTHOR]

Altered blood pressure and flow impact cardiac function during morphogenesis. How the arterial system supports cardiac morphogenesis after circulatory disruptions is not well characterized. We manipulated arterial flow via left atrial ligation (LAL) or arterial load v/a right vitelline artery ligation (VAL) in Hamburger-Hamilton (HH) stage 21 chick embryos. Embryos were reincubated for 1 h (HH21), 14 h (HH24) or 30 h (HH27). At each stage we measured simultaneous dorsal aortic blood pressure and flow, and calculated arterial compliance, impedance and hydraulic power. LAL acutely reduced stroke volume (Vs), cardiac output (Q) and hydraulic power. Arterial pressure was preserved by a compensatory increase in characteristic impedance and decrease in compliance. Impedance parameters and compliance normalized by HH24 and all parameters normalized by HH27. VAL acutely increased arterial resistance. Embryos maintained arterial pressure by decreasing Vs and Q. These parameters remained altered through HH27. In summary, despite the intervention, compensatory alterations in Vs and arterial resistance maintained arterial pressure and fraction of oscillatory power within a narrow range. These results suggest that the maintenance of arterial pressure and circulatory energy efficiency, but not arterial flow, is critical to embryogenesis. [ABSTRACT FROM AUTHOR]

Hemodynamic mechanical cues play a critical role in the early development and functional maturation of cardiomyocytes (CM). Therefore, tissue engineering approaches that incorporate immature CM intofunctional cardiac tissues capable of recovering or replacing damaged cardiac muscle require physiologically relevant environments to provide the appropriate mechanical cues. The goal of this work is to better understand the subcellular responses of immature cardiomyocytes using an in vitro cardiac cell culture model that realistically mimics in vivo mechanical conditions, including cyclical fluid flows, chamber pressures, and tissue strains that could be experienced by implanted cardiac tissues. Cardiomyocytes were cultured in a novel microfluidic cardiac cell culture model (CCCM) to achieve accurate replication of the mechanical cues experienced by ventricular CM. Day 10 chick embryonic ventricular CM (3.5 x 104 cell clusters per cell chamber) were cultured for 4 days in the CCCM under cyclic mechanical stimulation (10 mmHg, 8-15% stretch, 2 Hz frequency) and ventricular cells from the same embryo were cultured in a static condition for 4 days as controls. Additionally, ventricular cell suspensions and ventricular tissue from day 16 chick embryo were collected and analyzed for comparison with CCCM cultured CM. The gene expressions and protein synthesis of calcium handling proteins decreased significantly during the isolation process. Mechanical stimulation of the cultured CM using the CCCM resulted in an augmentation of gene expression and protein synthesis of calcium handling proteins compared to the 2D constructs cultured in the static conditions. Further, the CCCM conditioned 2D constructs have a higher beat rate and contractility response to isoproterenol. These results demonstrate that early mechanical stimulation of embryonic cardiac tissue is necessary for tissue proliferation and for protein synthesis of the calcium handling constituents required for tissue contractility. Thus, physiologic mechanical conditioning may be essential for generating functional cardiac patches for replacement of injured cardiac tissue. [ABSTRACT FROM AUTHOR]

A major challenge in cardiac tissue engineering is the delivery of hemodynamic mechanical cues that play a critical role in the early development and maturation of cardiomyocytes. Generation of functional cardiac tissue capable of replacing or augmenting cardiac function therefore requires physiologically relevant environments that can deliver complex mechanical cues for cardiomyocyte functional maturation. The goal of this work is the development and validation of a cardiac cell culture model (CCCM) microenvironment that accurately mimics pressure-volume changes seen in the left ventricle and to use this system to achieve cardiac cell maturation under conditions where mechanical loads such as pressure and stretch are gradually increased from the unloaded state to conditions seen in vivo. The CCCM platform, consisting of a cell culture chamber integrated within a flow loop was created to accomplish culture of 10 day chick embryonic ventricular cardiomyocytes subject to 4 days of stimulation (10 mmHg, ∼13% stretch at a frequency of 2 Hz). Results clearly show that CCCM conditioned cardiomyocytes accelerate cardiomyocyte structural and functional maturation in comparison to static unloaded controls as evidenced by increased proliferation, alignment of actin cytoskeleton, bundle-like sarcomeric a-actinin expression, higher pacing beat rate at lower threshold voltages, and increased shortening. These results confirm the CCCM microenvironment can accelerate immature cardiac cell structural and functional maturation for potential cardiac regenerative applications. [ABSTRACT FROM AUTHOR]