John F. Eberth

Time course of carotid artery growth and remodeling in response to altered pulsatility.

Elucidating early time courses of biomechanical responses by arteries to altered mechanical stimuli is paramount to understanding and eventually predicting long-term adaptations. In a previous study, we reported marked long-term (at 35-56 days) consequences of increased pulsatile hemodynamics on arterial structure and mechanics. Motivated by those findings, we focus herein on arterial responses over shorter periods (at 7, 10, and 14 days) following placement of a constrictive band on the aortic arch between the innominate and left carotid arteries of wild-type mice, which significantly increases pulsatility in the right carotid artery. We quantified hemodynamics in vivo using noninvasive ultrasound and measured wall properties and composition in vitro using biaxial mechanical testing and standard (immuno)histology. Compared with both baseline carotid arteries and left carotids after banding, right carotids after banding experienced a significant increase in both pulse pressure, which peaked at day 7, and a pulsatility index for velocity, which continued to rise over the 42-day study despite a transient increase in mean flow that peaked at day 7. Wall thickness and inner diameter also increased significantly in the right carotids, both peaking at day 14, with an associated marked early reduction in the in vivo axial stretch and a persistent decrease in smooth muscle contractility. Glycosaminoglycan content also increased within the wall, peaking at day 14, whereas increases in monocyte chemoattractant protein-1 activity and the collagen-to-elastin ratio continued to rise. These findings confirm that pulsatility is an important modulator of wall geometry, structure, and properties but reveal different early time courses for different microscopic and macroscopic metrics, presumably due to the separate degrees of influence of pressure and flow.

Consistent Biomechanical Phenotyping of Common Carotid Arteries from Seven Genetic, Pharmacological, and Surgical Mouse Models

The continuing lack of longitudinal histopathological and biomechanical data for human arteries in health and disease highlights the importance of studying the many genetic, pharmacological, and surgical models that are available in mice. As a result, there has been a significant increase in the number of reports on the biomechanics of murine arteries over the past decade, particularly for the common carotid artery. Whereas most of these studies have focused on wild-type controls or comparing controls vs. a single model of altered hemodynamics or vascular disease, there is a pressing need to compare results across many different models to understand more broadly the effects of genetic mutations, pharmacological treatments, or surgical alterations on the evolving hemodynamics and the microstructure and biomechanical properties of these vessels. This paper represents a first step toward this goal, that is, a biomechanical phenotyping of common carotid arteries from control mice and seven different mouse models that represent alterations in elastic fiber integrity, collagen remodeling, and smooth muscle cell functionality.

Altered Hemodynamics in the Embryonic Heart Affects Outflow Valve Development

Cardiac valve structure and function are primarily determined during early development. Consequently, abnormally-formed heart valves are the most common type of congenital heart defects. Several adult valve diseases can be backtracked to abnormal valve development, making it imperative to completely understand the process and regulation of heart valve development. Epithelial-to-mesenchymal transition (EMT) plays an important role in the development of heart valves. Though hemodynamics is vital to valve development, its role in regulating EMT is still unknown. In this study, intracardiac hemodynamics were altered by constricting the outflow tract (OFT)/ventricle junction (OVJ) of HH16–17 (Hamilton and Hamburger (HH) Stage 16–17) chicken embryos, ex ovo for 24 h. The constriction created an increase in peak and time-averaged centerline velocity along the OFT without changes to volumetric flow or heart rate. Computational fluid dynamics was used to estimate the level of increased spatially-averaged wall shear stresses on the OFT cushion from AMIRA reconstructions. OFT constriction led to a significant decrease in OFT cushion volume and the number of invaded mesenchyme in the OFT cushion. qPCR analysis revealed altered mRNA expression of a representative panel of genes, vital to valve development, in the OFT cushions from banded hearts. This study indicates the importance of hemodynamics in valve development.

Evolving biaxial mechanical properties of mouse carotid arteries in hypertension

Quantifying the time course of load-induced changes in arterial wall geometry, microstructure, and properties is fundamental to developing mathematical models of growth and remodeling. Arteries adapt to altered pressure and flow by modifying wall thickness, inner diameter, and axial length via marked cell and matrix turnover. To estimate particular biomaterial implications of such adaptations, we used a 4-fiber family constitutive relation to quantify passive biaxial mechanical behaviors of mouse carotid arteries 0 (control), 7-10, 10-14, or 35-56 days after an aortic arch banding surgery that increased pulse pressure and pulsatile flow in the right carotid artery. In vivo circumferential and axial stretches at mean arterial pressure were, for example, 11% and 26% lower, respectively, in hypertensive carotids 35-56 days after banding than in normotensive controls; this finding is consistent with observations that hypertension decreases distensibility. Interestingly, the strain energy W stored in the carotids at individual in vivo conditions was also less in hypertensive compared with normotensive carotids. For example, at 35-56 days after banding, W was 24%, 39%, and 47% of normal values at diastolic, mean, and systolic pressures, respectively. The energy stored during the cardiac cycle, W(sys)-W(dias), also tended to be less, but this reduction did not reach significance. When computed at normal in vivo values of biaxial stretch, however, W was well above normal for the hypertensive carotids. This net increase resulted from an overall increase in the collagen-related anisotropic contribution to W despite a decrease in the elastin-related isotropic contribution. The latter was consistent with observed decreases in the mass fraction of elastin.

The impact of flow-induced forces on the morphogenesis of the outflow tract

One percent of infants are born with congenital heart disease (CHD), which commonly involves outflow tract (OFT) defects. These infants often require complex surgeries, which are associated with long term adverse remodeling effects, and receive replacement valves with limited strength, biocompatibility, and growth capability. To address these problematic issues, researchers have carried out investigations in valve development and valve mechanics. A longstanding hypothesis is that flow-induced forces regulate fibrous valve development, however, the specific mechanisms behind this mechanotransduction remain unclear. The purpose of this study was to implement an in vitro system of outflow tract development to test the response of embryonic OFT tissues to fluid flow. A dynamic, three-dimensional bioreactor system was used to culture embryonic OFT tissue under different levels of flow as well as the absence of flow. In the absence of flow, OFT tissues took on a more primitive phenotype that is characteristic of early OFT cushion development where widely dispersed mesenchymal cells are surrounded by a sparse, disorganized extracellular matrix (ECM). Whereas OFT tissues subjected to physiologically matched flow formed compact mounds of cells, initated, fibrous ECM development, while prolonged supraphysiological flow resulted in abnormal tissue remodeling. This study indicates that both the timing and magnitude of flow alter cellular processes that determine if OFT precursor tissue undergoes normal or pathological development. Specifically, these experiments showed that flow-generated forces regulate the deposition and localization of fibrous ECM proteins, indicating that mechanosensitive signaling pathways are capable of driving pathological OFT development if flows are not ideal.

A mechanical argument for the differential performance of coronary artery grafts

Coronary artery bypass grafting (CABG) acutely disturbs the homeostatic state of the transplanted vessel making retention of graft patency dependent on chronic remodeling processes. The time course and extent to which remodeling restores vessel homeostasis will depend, in part, on the nature and magnitude of the mechanical disturbances induced upon transplantation. In this investigation, biaxial mechanical testing and histology were performed on the porcine left anterior descending artery (LAD) and analogs of common autografts, including the internal thoracic artery (ITA), radial artery (RA), great saphenous vein (GSV) and lateral saphenous vein (LSV). Experimental data were used to quantify the parameters of a structure-based constitutive model enabling prediction of the acute vessel mechanical response pre-transplantation and under coronary loading conditions. A novel metric Ξ was developed to quantify mechanical differences between each graft vessel in situ and the LAD in situ, while a second metric Ω compares the graft vessels in situ to their state under coronary loading. The relative values of these metrics among candidate autograft sources are consistent with vessel-specific variations in CABG clinical success rates with the ITA as the superior and GSV the inferior graft choices based on mechanical performance. This approach can be used to evaluate other candidate tissues for grafting or to aid in the development of synthetic and tissue engineered alternatives.

Comparison of Aortic Collagen Fiber Angle Distribution in Mouse Models of Atherosclerosis Using Second-Harmonic Generation (SHG) Microscopy

Characterization of collagen fiber angle distribution throughout the blood vessel wall provides insight into the mechanical behavior of healthy and diseased arteries and their capacity to remodel. Atherosclerotic plaque contributes to the overall mechanical behavior, yet little is known experimentally about how collagen fiber orientation is influenced by atherogenesis. We hypothesized that atherosclerotic lesion development, and the factors contributing to lesion development, leads to a shift in collagen fiber angles within the aorta. Second-harmonic generation microscopy was used to visualize the three-dimensional organization of collagen throughout the aortic wall and to examine structural differences in mice maintained on high-fat Western diet versus age-matched chow diet mice in a model of atherosclerosis. Image analysis was performed on thoracic and abdominal sections of the aorta from each mouse to determine fiber orientation, with the circumferential (0°) and blood flow directions (axial ±90°) as the two reference points. All measurements were used in a multiple regression analysis to determine the factors having a significant influence on mean collagen fiber angle. We found that mean absolute angle of collagen fibers is 43° lower in Western diet mice compared with chow diet mice. Mice on a chow diet have a mean collagen fiber angle of ±63°, whereas mice on a Western diet have a more circumferential fiber orientation (~20°). This apparent shift in absolute angle coincides with the development of extensive aortic atherosclerosis, suggesting that atherosclerotic factors contribute to collagen fiber angle orientation.

The perivascular environment along the vertebral artery governs segment-specific structural and mechanical properties.

The vertebral arteries (VAs) are anatomically divided into four segments (V1-V4), which cumulatively transport blood flow through neck and ultimately form the posterior circulation of the brain. The vital physiological function of these conduit vessels depends on their geometry, composition and mechanical properties, all of which may vary among the defined arterial segments. Despite their significant role in blood circulation and susceptibility to injury, few studies have focused on characterizing the mechanical properties of VAs, and none have investigated the potential for segmental variation that could arise due to distinct perivascular environments. In this study, we compare the passive mechanical response of the central, juxtaposed arterial segments of porcine VAs (V2 and V3) via inflation-extension mechanical testing. Obtained experimental data and histological measures of arterial wall composition were used to adjust parameters of structure-motivated constitutive models that quantify the passive mechanical properties of each arterial segment and enable prediction of wall stress distributions under physiologic loads and boundary conditions. Our findings reveal significant segmental differences in the arterial wall geometry and structure. Nevertheless, similar wall stress distributions are predicted in these neighboring arterial segments if calculations account for their specific perivascular environments. These findings allow speculation that segmental differences in wall structure and geometry are a consequence of a previously introduced principle of optimal operation of arteries, which ensures effective bearing of physiological load and a favorable mechanical environment for mechanosensitive vascular smooth muscle cells. STATEMENT OF SIGNIFICANCE Among the numerous biomechanical investigations devoted to conduit blood vessels, only a few deal with vertebral arteries. While these studies provide useful information that describes the vessel mechanical response, they do not enable identification of a constitutive formulation of the mechanical properties of the vessel wall. This is an important distinction, as a constitutive material model is required to calculate the local stress environment of mechanosensitive vascular cells and fully understand the mechanical implications of both vascular injury and clinical intervention. Moreover, segmental differences in the mechanical properties of the vertebral arteries could be used to discriminate among distinct modes of injury and disease etiologies.

Contractile Smooth Muscle and Active Stress Generation in Porcine Common Carotids

The mechanical response of intact blood vessels to applied loads can be delineated into passive and active components using an isometric decomposition approach. Whereas the passive response is due predominantly to the extracellular matrix (ECM) proteins and amorphous ground substance, the active response depends on the presence of smooth muscle cells (SMCs) and the contractile machinery activated within those cells. To better understand determinants of active stress generation within the vascular wall, we subjected porcine common carotid arteries (CCAs) to biaxial inflation-extension testing under maximally contracted or passive SMC conditions and semiquantitatively measured two known markers of the contractile SMC phenotype: smoothelin and smooth muscle-myosin heavy chain (SM-MHC). Using isometric decomposition and established constitutive models, an intuitive but novel correlation between the magnitude of active stress generation and the relative abundance of smoothelin and SM-MHC emerged. Our results reiterate the importance of stretch-dependent active stress generation to the total mechanical response. Overall these findings can be used to decouple the mechanical contribution of SMCs from the ECM and is therefore a powerful tool in the analysis of disease states and potential therapies where both constituent are altered.

Biofabrication of Dynamic, 3-Dimensional, In vitro Models of Disease

1. University of South Carolina, School of Medicine, Biomedical Sciences Dept., Greenville, SC USA 2. University of South Carolina, Biomedical Engineering Program, Columbia, SC USA 3. University of Michigan, Civil and Environmental Engineering Dept., Ann Arbor, MI USA 4. University of South Carolina, School of Medicine, Dept. of Cell Biology and Anatomy. Columbia, SC USA. 5. University of South Carolina, School of Medicine, Columbia, SC USA 6. University of South Carolina, School of Medicine. Instrument Resource Facility, Columbia, SC USA. 7. University of South Carolina, School of Medicine, Dept. of Surgery. Columbia, SC USA. 8. Medical University of South Carolina, Dept. of Surgery, Charleston, SC USA