Unique Morphology and Focal Adhesion Development of Valvular Endothelial Cells in Static and Fluid Flow Environments

Arteriosclerosis, Thrombosis, and Vascular Biology. Aug, 2004  |  Pubmed ID: 15117733

The influence of mechanical forces on cell function has been well documented for many different cell types. Endothelial cells native to the aortic valve may play an important role in mediating tissue responses to the complex fluid environment, and may therefore respond to fluid flow in a different manner than more characterized vascular endothelial cells.

Porcine Aortic Valve Interstitial Cells in Three-dimensional Culture: Comparison of Phenotype with Aortic Smooth Muscle Cells

The Journal of Heart Valve Disease. May, 2004  |  Pubmed ID: 15222296

Recent heart valve tissue engineering efforts have involved populating scaffolds with cells isolated from vascular sources, though it is unclear whether cells from valvular origins behave similarly to vascular cells. The study aim was to compare the phenotype of porcine aortic valve interstitial cells (PAVICs) and porcine aortic smooth muscle cells (PASMCs) in two-dimensional cultures and within three-dimensional (3D) collagen gels.

Transcriptional Profiles of Valvular and Vascular Endothelial Cells Reveal Phenotypic Differences: Influence of Shear Stress

Arteriosclerosis, Thrombosis, and Vascular Biology. Jan, 2006  |  Pubmed ID: 16293796

The similarities between valvular and vascular lesions suggest pathological initiation mediated through endothelium, but the role of hemodynamics in valvular endothelial biology is poorly understood.

Valvular Endothelial Cells Regulate the Phenotype of Interstitial Cells in Co-culture: Effects of Steady Shear Stress

Tissue Engineering. Apr, 2006  |  Pubmed ID: 16674302

Valvular endothelial cells interact with interstitial cells in a complex hemodynamic and mechanical environment to maintain leaflet tissue integrity. The precise roles of each cell type are difficult to ascertain in a controlled manner in vivo. The objective of this study was to develop a three-dimensional aortic valve leaflet model, comprised of valvular endothelium and interstitial cells, and determine the cellular responses to imposed lumenal fluid flow. Two leaflet models were created using type I collagen hydrogels. Model 1 contained 1 million/mL porcine aortic valve interstitial cells (PAVICs). Model 2 added a seeding of the lumenal surface of Model 1 with approximately 50,000/cm(2) porcine aortic valve endothelial cells (PAVECs). Both leaflet models were exposed to 20 dynes/cm(2) steady shear for up to 96 h, with static constructs serving as controls. Endothelial cell alignment, matrix production, and cell phenotype were monitored. The results indicate that PAVECs align perpendicularly to flow similar to 2D culture. We report that PAVICs in model 1 express vimentin strongly and alpha-smooth-muscle actin (SMA) to a lesser extent, but SMA expression is increased by shear stress, particularly near the lumenal surface. Model 1 constructs increase in cell number, maintain protein levels, but lose glycosaminoglycans in response to shear. Co-culture with PAVECs (Model 2) modulates these responses in both static and flow environments, resulting in PAVIC phenotype that is more similar to the native condition. PAVECs stimulated a decrease in PAVIC proliferation, an increase in protein synthesis with shear stress, and reduced the loss of glycosaminoglycans with flow. Additionally, PAVECs stimulated PAVIC differentiation to a more quiescent phenotype, defined by reduced expression of SMA. These results suggest that valvular endothelial cells are necessary to properly regulate interstitial cell phenotype and matrix synthesis. Additionally, we show that tissue-engineered models can be used to discover and understand complex biomechanical relationships between cells that interact in vivo.

Equibiaxial Strain Stimulates Fibroblastic Phenotype Shift in Smooth Muscle Cells in an Engineered Tissue Model of the Aortic Wall

Biomaterials. Oct, 2006  |  Pubmed ID: 16806457

Many cells in the body reside in a complex three-dimensional (3D) environment stimulated by mechanical force. In vitro bioreactor systems have greatly improved our understanding of the mechanisms behind cell mechanotransduction. Current systems to impose strain in vitro are limited either by the lack of uniform strain profile or inability to strain 3D engineered tissues. In this study, we present a system capable of generating cyclic equibiaxial strain to an engineered vascular wall model. Type I collagen hydrogels populated with rat aortic smooth muscle cells (RASMCs) were created either as a compacting disk or constrained hemisphere. Both models were adhered to silicone membranes precoated with collagen I, fibronectin, or Cell-Tak and assayed for adhesion characteristics. The best performing model was then exposed to 48 h of 10% strain at 1Hz to simulate wall strain profiles found in vascular aneurysms, with static cultures serving as controls. The finite strain profile at the level of the membrane and the free surface of the construct was quantified using microbeads. The results indicate that the hemisphere model adhered with Cell-Tak had the most stable adhesion, followed by fibronectin and collagen I. Disk models did not adhere well under any coating condition. Uniform strain propagation was possible up to a maximum area strain of 20% with this system. RASMC responded to 10% equibiaxial strain by becoming less elongated, and immunohistochemistry suggested that stretched RASMC shifted to a more synthetic phenotype in comparison to static controls. These results suggest that equibiaxial strain may induce smooth muscle cell differentiation. We conclude that this system is effective in stimulating cells with cyclic equibiaxial strain in 3D cultures, and can be applied to a variety of biomaterial and tissue engineering applications.

The Next Frontier in Cardiovascular Developmental Biology--an Integrated Approach to Adult Disease?

Nature Clinical Practice. Cardiovascular Medicine. Feb, 2007  |  Pubmed ID: 17245398

Valvular Endothelial Cells and the Mechanoregulation of Valvular Pathology

Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. Aug, 2007  |  Pubmed ID: 17569641

Endothelial cells are critical mediators of haemodynamic forces and as such are important foci for initiation of vascular pathology. Valvular leaflets are also lined with endothelial cells, though a similar role in mechanosensing has not been demonstrated. Recent evidence has shown that valvular endothelial cells respond morphologically to shear stress, and several studies have implicated valvular endothelial dysfunction in the pathogenesis of disease. This review seeks to combine what is known about vascular and valvular haemodynamics, endothelial response to mechanical stimuli and the pathogenesis of valvular diseases to form a hypothesis as to how mechanical stimuli can initiate valvular endothelial dysfunction and disease progression. From this analysis, it appears that inflow surface-related bacterial/thrombotic vegetative endocarditis is a high shear-driven endothelial denudation phenomenon, while the outflow surface with its related calcific/atherosclerotic degeneration is a low/oscillatory shear-driven endothelial activation phenomenon. Further understanding of these mechanisms may help lead to earlier diagnostic tools and therapeutic strategies.

Quantitative Volumetric Analysis of Cardiac Morphogenesis Assessed Through Micro-computed Tomography

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Mar, 2007  |  Pubmed ID: 17013892

We present a method to generate quantitative embryonic cardiovascular volumes at extremely high resolution without tissue shrinkage using micro-computed tomography (Micro-CT). A CT dense polymer (Microfil, Flow Tech, Inc.) was used to perfuse avian embryonic hearts from Hamburger and Hamilton stage (HH) 15 through HH36, which solidified to create a cast within the luminal space. Hearts were then scanned at 10.5 mum(3) voxel resolution using a VivaCT scanner, digital slices were contoured for regions of interest, and computational analysis was conducted to quantify morphogenetic parameters. The three-dimensional morphology was compared with that of scanning electron microscopy (SEM) images and serial section reconstruction of similarly staged hearts. We report that Microfil-perfused hearts swelled to maximum end-diastolic volume with negligible shrinking after polymerization. Comparison to SEM revealed good agreement of cardiac chamber proportions and intracardiac tissue structures (i.e., valves and septa) at the stages of development assessed. Quantification of changes in chamber volume over development revealed several notable results that confirm earlier hypotheses. Heart chamber volumes grow over two orders of magnitude during the 1-week developmental period analyzed. The atrioventricular canal comprised a significant proportion of the early heart volume. While left atrium/left ventricular volume ratios approached 1 in later development, right atrium/right ventricle ratios increase to over 2.5. Quantification of trabeculation patterns confirmed that the right and left ventricles are similarly trabeculated before HH27, after which the right ventricle became quantitatively coarser than that of the left ventricle. These results demonstrate that Micro-CT can be used to image and quantify cardiovascular structures during development.

Periostin Promotes Atrioventricular Mesenchyme Matrix Invasion and Remodeling Mediated by Integrin Signaling Through Rho/PI 3-kinase

Developmental Biology. Feb, 2007  |  Pubmed ID: 17070513

Recent evidence suggests that extracellular matrix components may play a signaling role in embryonic valve development. We have previously identified the spatiotemporal expression patterns of periostin in developing valves, but its function during this process is largely unknown. To evaluate the functional role periostin plays during valvulogenesis, two separate three-dimensional culture assay systems, which model chick atrioventricular cushion development, were employed. These assays demonstrated that cushion mesenchymal cells adhered and spread on purified periostin in a dose-responsive manner, similar to collagen I and fibronectin via alpha(v)beta(3) and beta(1) integrin pairs. Periostin overexpression resulted in enhanced mesenchyme invasion through 3D collagen gels and increased matrix compaction. This invasion was dependent on alpha(v)beta(3) more than beta(1) integrin signaling, and was mediated differentially by Rho kinase and PI 3-kinase. Both matrix invasion and compaction were associated with a colocalization of periostin and beta(1) integrin expression to migratory cell phenotype in both surface and deep cells. The Rho/PI 3-kinase pathway also differentially mediated matrix compaction. Both Rho and PI 3-kinase were involved in normal cushion mesenchyme matrix compaction, but only PI 3-kinase was required for the enhanced matrix compaction due to periostin. Taken together, these results highlight periostin as a mediator of matrix remodeling by cushion mesenchyme towards a mature valve structure.

Transitions in Early Embryonic Atrioventricular Valvular Function Correspond with Changes in Cushion Biomechanics That Are Predictable by Tissue Composition

Circulation Research. May, 2007  |  Pubmed ID: 17478728

Endocardial cushions are critical to maintain unidirectional blood flow under constantly increasing hemodynamic forces, but the interrelationship between endocardial cushion structure and the mechanics of atrioventricular junction function is poorly understood. Atrioventricular (AV) canal motions and blood velocities of embryonic chicks at Hamburger and Hamilton (HH) stages 17, 21, and 25 were quantified using ultrasonography. Similar to the embryonic zebrafish heart, the HH17 AV segment functions like a suction pump, with the cushions expanding in a wave during peak myocardial contraction and becoming undetectable during the relaxation phase. By HH25, the AV canal contributes almost nothing to the piston-like propulsion of blood, but the cushions function as stoppers apposing blood flow with near constant thickness. Using a custom built mesomechanical testing system, we quantified the nonlinear pseudoelastic biomechanics of developing AV cushions, and found that both AV cushions increased in effective modulus between HH17 and HH25. Enzymatic digestion of major structural constituent collagens or glycosaminoglycans resulted in distinctly different stress-strain curves suggestive of their individual contributions. Mixture theory using histologically determined volume fractions of cells, collagen, and glycosaminoglycans showed good prediction of cushion material properties regardless of stage and cushion position. These results have important implications in valvular development, as biomechanics may play a larger role in stimulating valvulogenic events than previously thought.

Valvulogenesis: the Moving Target

Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. Aug, 2007  |  Pubmed ID: 17569640

Valvulogenesis is an extremely complex process by which a fragile gelatinous matrix is populated and remodelled during embryonic development into thin fibrous leaflets capable of maintaining unidirectional flow over a lifetime. This process occurs during exposure to constantly changing haemodynamic forces, with a success rate of approximately 99%. Defective valvulogenesis results in impaired cardiac function and lifelong complications. This review integrates what is known about the roles of genetics and mechanics in the development of valves and how changes in either result in impaired morphogenesis. It is hoped that appropriate developmental cues and phenotypic endpoints could help engineers and clinicians in their efforts to regenerate living valve alternatives.

Neonatal and Adult Cardiovascular Pathophysiological Remodeling and Repair: Developmental Role of Periostin

Annals of the New York Academy of Sciences. Mar, 2008  |  Pubmed ID: 18375575

The neonatal heart undergoes normal hypertrophy or compensation to complete development and adapt to increased systolic pressures. Hypertrophy and increased neonatal wall stiffness are associated with a doubling of the number of fibroblasts and de novo formation of collagen. Normal postnatal remodeling is completed within 3-4 weeks after birth but can be rekindled in adult life in response to environmental signals that lead to pathological hypertrophy, fibrosis, and heart failure. The signals that trigger fibroblast and collagen formation (fibrosis) as well as the origin and differentiation of the cardiac fibroblast lineage are not well understood. Using mice studies and a single-cell engraftment model, we have shown that cardiac fibroblasts are derived from two extracardiac sources: the embryonic proepicardial organ and the recruitment of circulating bone marrow cells of hematopoietic stem cell origin. Periostin, a matricellular protein, is normally expressed in differentiating fibroblasts but its expression is elevated several fold in pathological remodeling and heart failure. Our hypothesis that periostin is profibrogenic (i.e., it promotes differentiation of progenitor mesenchymal cells into fibroblasts and their secretion and compaction of collagen) was tested using isolated and cultured embryonic, neonatal, and adult wild-type and periostin-null, nonmyocyte populations. Our findings indicate that abrogation of periostin by targeted gene deletion inhibits differentiation of nonmyocyte progenitor cells or permits misdirection into a cardiomyocyte lineage. However, if cultured with periostin or forced to express periostin, they became fibroblasts. Periostin plays a significant role in promoting fibrogenesis residual stress, and tensile testings indicated that periostin played an essential regulatory role in maintaining the biomechanical properties of the adult myocardium. These findings indicate that periostin is a profibrogenic matricellular protein that promotes collagen fibrogenesis, inhibits differentiation of progenitor cells into cardiomyocytes, and is essential for maintaining the biomechanical properties of the adult myocardium.

Mechanobiology of the Aortic Heart Valve

The Journal of Heart Valve Disease. Jan, 2008  |  Pubmed ID: 18365571

The aortic heart valve is a complex and sophisticated structure that functions in a mechanically challenging environment. With each cardiac cycle, blood flow exerts shear stresses, bending stress and tensile and compressive forces on the valve tissue. These forces determine a plethora of biological responses, including gene expression, protein activation and cell phenotype. Consequently, mechanical forces may influence valve remodeling or pathological changes. Understanding the mechanobiology of heart valves is a vast task. Herein, some of the recent studies that have increased current knowledge of endothelial and interstitial cell interactions with physical forces are examined. Additionally, experimental co-culture models are described that are being developed to further improve the understanding of endothelial-interstitial cell interactions. Finally, the means by which organ culture systems are being utilized to study heart valve biology, thereby providing a complementary approach to in vivo experimentation, are described.

Cyclic Strain Regulates Pro-inflammatory Protein Expression in Porcine Aortic Valve Endothelial Cells

The Journal of Heart Valve Disease. Sep, 2008  |  Pubmed ID: 18980092

The endothelium of diseased heart valves is known to express the adhesion molecules VCAM-1, ICAM-1 and E-selectin, while healthy valves lack these pro-inflammatory proteins. The study aim was to determine if mechanical forces were responsible for the pro-inflammatory reaction in aortic valve endothelial cells.

Two-photon Microscopy-guided Femtosecond-laser Photoablation of Avian Cardiogenesis: Noninvasive Creation of Localized Heart Defects

American Journal of Physiology. Heart and Circulatory Physiology. Nov, 2010  |  Pubmed ID: 20709864

Embryonic heart formation is driven by complex feedback between genetic and hemodynamic stimuli. Clinical congenital heart defects (CHD), however, often manifest as localized microtissue malformations with no underlying genetic mutation, suggesting that altered hemodynamics during embryonic development may play a role. An investigation of this relationship has been impaired by a lack of experimental tools that can create locally targeted cardiac perturbations. Here we have developed noninvasive optical techniques that can modulate avian cardiogenesis to dissect relationships between alterations in mechanical signaling and CHD. We used two-photon excited fluorescence microscopy to monitor cushion and ventricular dynamics and femtosecond pulsed laser photoablation to target micrometer-sized volumes inside the beating chick hearts. We selectively photoablated a small (∼100 μm radius) region of the superior atrioventricular (AV) cushion in Hamburger-Hamilton 24 chick embryos. We quantified via ultrasound that the disruption causes AV regurgitation, which resulted in a venous pooling of blood and severe arterial constriction. At 48 h postablation, quantitative X-ray microcomputed tomography imaging demonstrated stunted ventricular growth and pronounced left atrial dilation. A histological analysis demonstrated that the laser ablation produced defects localized to the superior AV cushion: a small quasispherical region of cushion tissue was completely obliterated, and the area adjacent to the myocardial wall was less cellularized. Both cushions and myocardium were significantly smaller than sham-operated controls. Our results highlight that two-photon excited fluorescence coupled with femtosecond pulsed laser photoablation should be considered a powerful tool for studying hemodynamic signaling in cardiac morphogenesis through the creation of localized microscale defects that may mimic clinical CHD.

Transforming Growth Factor β, Bone Morphogenetic Protein, and Vascular Endothelial Growth Factor Mediate Phenotype Maturation and Tissue Remodeling by Embryonic Valve Progenitor Cells: Relevance for Heart Valve Tissue Engineering

Tissue Engineering. Part A. Nov, 2010  |  Pubmed ID: 20629541

Despite years of research, limited understanding of heart valve cell and tissue biology remains a key impediment to valvular tissue engineering progress. Heart valves rapidly evolve structural and cellular composition naturally during embryonic development, which suggests that mimicking these signaling events could advance engineered valve tissue research. Many inductive factors participate in the initial endocardial to mesenchymal transformation event necessary to form the prevalvular cushion, but far less is known about the regulation of cushion remodeling into fibrous leaflets and the associated maturation of valvular progenitors into fibroblasts. In this study, we combine in vitro three-dimensional tissue-engineered models of embryonic valvular remodeling with in vivo analysis to determine the roles of three prominent growth factors during avian mitral valvulogenesis. We show that transforming growth factor-β3 (TGFβ3), bone morphogenetic protein 2 (BMP2), and vascular endothelial growth factor A (VEGFA) are expressed in spatiotemporally distinct patterns and at significantly different levels within remodeling embryonic valves in vivo. We then establish dose-dependent functional roles for each growth factor in 3D cultured embryonic valve progenitor cells. TGFβ3 induced cell migration, invasion, and matrix condensation; BMP2 induced invasion. VEGFA inhibited invasion but increased migration. Finally, we determine that TGFβ3 induced myofibroblastic differentiation in a dose-dependent manner, whereas VEGFA and BMP2 did not. Collectively, these findings frame a naturally derived blueprint for controlling valvulogenic remodeling and phenotype maturation, which can be integrated into clinically needed regenerative strategies for heart valve disease and to accelerate the development of engineered tissue valves.

Hemodynamic Patterning of the Avian Atrioventricular Valve

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jan, 2011  |  Pubmed ID: 21181939

In this study, we develop an innovative approach to rigorously quantify the evolving hemodynamic environment of the atrioventricular (AV) canal of avian embryos. Ultrasound generated velocity profiles were imported into Micro-Computed Tomography generated anatomically precise cardiac geometries between Hamburger-Hamilton (HH) stages 17 and 30. Computational fluid dynamic simulations were then conducted and iterated until results mimicked in vivo observations. Blood flow in tubular hearts (HH17) was laminar with parallel streamlines, but strong vortices developed simultaneous with expansion of the cushions and septal walls. For all investigated stages, highest wall shear stresses (WSS) are localized to AV canal valve-forming regions. Peak WSS increased from 19.34 dynes/cm(2) at HH17 to 287.18 dynes/cm(2) at HH30, but spatiotemporally averaged WSS became 3.62 dynes/cm(2) for HH17 to 9.11 dynes/cm(2) for HH30. Hemodynamic changes often preceded and correlated with morphological changes. These results establish a quantitative baseline supporting future hemodynamic analyses and interpretations.

Quantitative Three-dimensional Analysis of Embryonic Chick Morphogenesis Via Microcomputed Tomography

Anatomical Record (Hoboken, N.J. : 2007). Jan, 2011  |  Pubmed ID: 21207522

Embryonic development is a remarkably complex and rapidly evolving morphogenetic process. Although many of the early patterning events have been well described, understanding the anatomical changes at later stages where clinically relevant malformations are more likely to be survivable has been limited by the lack of quantitative 3D imaging tools. Microcomputed tomography (Micro-CT) has emerged as a powerful tool for embryonic imaging, but a quantitative analysis of organ and tissue growth has not been conducted. In this study, we present a simple method for acquiring highly detailed, quantitative 3D datasets of embryonic chicks with Micro-CT. Embryos between 4 and 12 days (HH23 and HH40) were labeled with osmium tetroxide (OT), which revealed highly detailed soft tissue anatomy when scanned at 25 μm resolution. We demonstrate tissue boundary and inter-tissue contrast fidelity in virtual 2D sections are quantitatively and qualitatively similar to those of histological sections. We then establish mathematical relationships for the volumetric growth of heart, limb, eye, and brain during this period of development. We show that some organs exhibit constant exponential growth (eye and heart), whereas others contained multiple phases of growth (forebrain and limb). Furthermore, we show that cardiac myocardial volumetric growth differs in a time and chamber specific manner. These results demonstrate Micro-CT is a powerful technique for quantitative imaging of embryonic growth. The data presented here establish baselines from which to compare the effects of genetic or experimental perturbations. Quantifying subtle differences in morphogenesis is increasingly important as research focuses on localized and conditional effects.

Aortic Valve Disease and Treatment: the Need for Naturally Engineered Solutions

Advanced Drug Delivery Reviews. Apr, 2011  |  Pubmed ID: 21281685

The aortic valve regulates unidirectional flow of oxygenated blood to the myocardium and arterial system. The natural anatomical geometry and microstructural complexity ensures biomechanically and hemodynamically efficient function. The compliant cusps are populated with unique cell phenotypes that continually remodel tissue for long-term durability within an extremely demanding mechanical environment. Alteration from normal valve homeostasis arises from genetic and microenvironmental (mechanical) sources, which lead to congenital and/or premature structural degeneration. Aortic valve stenosis pathobiology shares some features of atherosclerosis, but its final calcification endpoint is distinct. Despite its broad and significant clinical significance, very little is known about the mechanisms of normal valve mechanobiology and mechanisms of disease. This is reflected in the paucity of predictive diagnostic tools, early stage interventional strategies, and stagnation in regenerative medicine innovation. Tissue engineering has unique potential for aortic valve disease therapy, but overcoming current design pitfalls will require even more multidisciplinary effort. This review summarizes the latest advancements in aortic valve research and highlights important future directions.

Quantitative Three-dimensional Imaging of Live Avian Embryonic Morphogenesis Via Micro-computed Tomography

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Aug, 2011  |  Pubmed ID: 21761480

Many clinically relevant congenital malformations arise during mid to late embryonic stages. This period is challenging to image quantitatively in live embryos, necessitating the use of multiple specimens with increased experimental variability. Here we establish X-ray and blood-pool computed tomography (CT) contrast agent toxicity and teratogenesis thresholds for 3D Micro-CT imaging of live avian embryos. Day 4 chick embryos micro-injected with Visipaque™ (VP) developed for an additional 6 days without defect. X-ray radiation up to 798 mGy was nontoxic. Peak average contrast of 1,060 HU occurred within 1 hr of imaging at 50 μm resolution. VP-enhanced contrast persisted past 24 hr with delayed accumulation in the allantois. Regional volumes of VP-injected embryos were statistically identical to those of fixed embryos perfused with osmium tetroxide. We further quantified longitudinal volumetric morphogenesis of the allantois over 30 hr. These results demonstrate the safety and efficacy of contrast enhanced quantitative micro-CT imaging for live embryos.

Inflammatory Regulation of Valvular Remodeling: the Good(?), the Bad, and the Ugly

International Journal of Inflammation. 2011  |  Pubmed ID: 21792386

Heart valve disease is unique in that it affects both the very young and very old, and does not discriminate by financial affluence, social stratus, or global location. Research over the past decade has transformed our understanding of heart valve cell biology, yet still more remains unclear regarding how these cells respond and adapt to their local microenvironment. Recent studies have identified inflammatory signaling at nearly every point in the life cycle of heart valves, yet its role at each stage is unclear. While the vast majority of evidence points to inflammation as mediating pathological valve remodeling and eventual destruction, some studies suggest inflammation may provide key signals guiding transient adaptive remodeling. Though the mechanisms are far from clear, inflammatory signaling may be a previously unrecognized ally in the quest for controlled rapid tissue remodeling, a key requirement for regenerative medicine approaches for heart valve disease. This paper summarizes the current state of knowledge regarding inflammatory mediation of heart valve remodeling and suggests key questions moving forward.

Quantification of Embryonic Atrioventricular Valve Biomechanics During Morphogenesis

Journal of Biomechanics. Dec, 2011  |  Pubmed ID: 22169154

Tissue assembly in the developing embryo is a rapid and complex process. While much research has focused on genetic regulatory machinery, understanding tissue level changes such as biomechanical remodeling remains a challenging experimental enigma. In the particular case of embryonic atrioventricular valves, micro-scale, amorphous cushions rapidly remodel into fibrous leaflets while simultaneously interacting with a demanding mechanical environment. In this study we employ two microscale mechanical measurement systems in conjunction with finite element analysis to quantify valve stiffening during valvulogenesis. The pipette aspiration technique is compared to a uniaxial load deformation, and the analytic expression for a uniaxially loaded bar is used to estimate the nonlinear material parameters of the experimental data. Effective modulus and strain energy density are analyzed as potential metrics for comparing mechanical stiffness. Avian atrioventricular valves from globular Hamburger-Hamilton stages HH25-HH34 were tested via the pipette method, while the planar HH36 leaflets were tested using the deformable post technique. Strain energy density between HH25 and HH34 septal leaflets increased 4.6±1.8 fold (±SD). The strain energy density of the HH36 septal leaflet was four orders of magnitude greater than the HH34 pipette result. Our results establish morphological thresholds for employing the micropipette aspiration and deformable post techniques for measuring uniaxial mechanical properties of embryonic tissues. Quantitative biomechanical analysis is an important and underserved complement to molecular and genetic experimentation of embryonic morphogenesis.

Cardiac Developmental Toxicity

Birth Defects Research. Part C, Embryo Today : Reviews. Dec, 2011  |  Pubmed ID: 22271678

Congenital heart disease (CHD) is a highly prevalent problem with mostly unknown origins. Many cases of CHD likely involve an environmental exposure coupled with genetic susceptibility, but practical and ethical considerations make nongenetic causes of CHD difficult to assess in humans. The development of the heart is highly conserved across all vertebrate species, making animal models an excellent option for screening potential cardiac teratogens. This review will discuss exposures known to cause cardiac defects, stages of heart development that are most sensitive to teratogen exposure, benefits and limitations of animal models of cardiac development, and future considerations for cardiac developmental toxicity research. Birth Defects Research (Part C) 93:291-297, 2011. © 2012 Wiley Periodicals, Inc.

Quantitative Three-dimensional Analysis of Embryonic Chick Morphogenesis Via Microcomputed Tomography

Anatomical Record (Hoboken, N.J. : 2007). Jan, 2011  |  Pubmed ID: 21157911

Embryonic development is a remarkably complex and rapidly evolving morphogenetic process. Although many of the early patterning events have been well described, understanding the anatomical changes at later stages where clinically relevant malformations are more likely to be survivable has been limited by the lack of quantitative 3D imaging tools. Microcomputed tomography (Micro-CT) has emerged as a powerful tool for embryonic imaging, but a quantitative analysis of organ and tissue growth has not been conducted. In this study, we present a simple method for acquiring highly detailed, quantitative 3D datasets of embryonic chicks with Micro-CT. Embryos between 4 and 12 days (HH23 and HH40) were labeled with osmium tetroxide (OT), which revealed highly detailed soft tissue anatomy when scanned at 25 microm resolution. We demonstrate tissue boundary and inter-tissue contrast fidelity in virtual 2D sections are quantitatively and qualitatively similar to those of histological sections. We then establish mathematical relationships for the volumetric growth of heart, limb, eye, and brain during this period of development. We show that some organs exhibit constant exponential growth (eye and heart), whereas others contained multiple phases of growth (forebrain and limb). Furthermore, we show that cardiac myocardial volumetric growth differs in a time and chamber specific manner. These results demonstrate Micro-CT is a powerful technique for quantitative imaging of embryonic growth. The data presented here establish baselines from which to compare the effects of genetic or experimental perturbations. Quantifying subtle differences in morphogenesis is increasingly important as research focuses on localized and conditional effects.

Cyclic Strain Anisotropy Regulates Valvular Interstitial Cell Phenotype and Tissue Remodeling in Three-dimensional Culture

Acta Biomaterialia. Jan, 2012  |  Pubmed ID: 22281945

Many planar connective tissues exhibit complex anisotropic matrix fiber arrangements that are critical to their biomechanical function. This organized structure is created and modified by resident fibroblasts in response to mechanical forces in their environment. The directionality of applied strain fields changes dramatically during development, aging, and disease, but the specific effect of strain direction on matrix remodeling is less clear. Current mechanobiological inquiry of planar tissues is limited to equibiaxial or uniaxial stretch, which inadequately simulates many in vivo environments. In this study, we implement a novel bioreactor system to demonstrate the unique effect of controlled anisotropic strain on fibroblast behavior in three-dimensional (3-D) engineered tissue environments, using aortic valve interstitial fibroblast cells as a model system. Cell seeded 3-D collagen hydrogels were subjected to cyclic anisotropic strain profiles maintained at constant areal strain magnitude for up to 96h at 1Hz. Increasing anisotropy of biaxial strain resulted in increased cellular orientation and collagen fiber alignment along the principal directions of strain and cell orientation was found to precede fiber reorganization. Cellular proliferation and apoptosis were both significantly enhanced under increasing biaxial strain anisotropy (P<0.05). While cyclic strain reduced both vimentin and alpha-smooth muscle actin compared to unstrained controls, vimentin and alpha-smooth muscle actin expression increased with strain anisotropy and correlated with direction (P<0.05). Collectively, these results suggest that strain field anisotropy is an independent regulator of fibroblast cell phenotype, turnover, and matrix reorganization, which may inform normal and pathological remodeling in soft tissues.