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In JoVE (2)
- Mekanisk testing av Mus carotis: fra nyfødte til voksen
- Måling Venstre ventrikkels Trykket i sen Embryonale og Neonatal Mus
Other Publications (16)
- Annals of Biomedical Engineering
- Journal of Biomechanical Engineering
- Annals of Biomedical Engineering
- American Journal of Physiology. Heart and Circulatory Physiology
- Journal of Cellular Physiology
- Biomechanics and Modeling in Mechanobiology
- American Journal of Physiology. Heart and Circulatory Physiology
- Birth Defects Research. Part C, Embryo Today : Reviews
- Circulation Research
- Physiological Reviews
- Arteriosclerosis, Thrombosis, and Vascular Biology
- American Journal of Physiology. Heart and Circulatory Physiology
- Biomechanics and Modeling in Mechanobiology
- American Journal of Physiology. Heart and Circulatory Physiology
- Journal of Biomechanical Engineering
- Journal of Cardiovascular Translational Research
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Articles by Jessica E. Wagenseil in JoVE
JoVE Bioengineering
Mekanisk testing av Mus carotis: fra nyfødte til voksen
Department of Biomedical Engineering, Saint Louis University
JoVE Bioengineering
Måling Venstre ventrikkels Trykket i sen Embryonale og Neonatal Mus
1Department of Biomedical Engineering, Saint Louis University, 2Department of Internal Medicine, Washington University School of Medicine
Måling venstre ventrikkel trykk (LV) i embryonale og neonatal mus er beskrevet. Trykket måles ved å sette inn en nål tilkoblet en væskefylt svingeren i LV i henhold til ultralyd veiledning. Det må tas for å opprettholde normal hjertefunksjon under forsøksprotokoll.
Other articles by Jessica E. Wagenseil on PubMed
Mechanical Properties of Dilated Human Ascending Aorta
Dilation of the ascending aorta, associated with Marfan Syndrome, bicuspid aortic valve, or advanced age, may lead to aortic dissection and rupture. Mathematical models can be used to assess the relative importance of increased wall stresses and decreased strength in these mechanical failures. To obtain needed inputs for such models, mechanical properties of dilated human ascending aorta were measured in vitro. Specimens for opening angle, biaxial elastic, and uniaxial circumferential strength tests were cut from excised tissue obtained from 54 patients (age 18-81 years) undergoing elective aortic graft replacement surgery. Opening angle was significantly greater in patients older than 50 years (262 degrees + 76 degrees, n = 21) compared to younger patients (202 degrees +/- 70 degrees, n = 13). All biaxial elastic specimens (n = 40) exhibited nonlinear stress-strain behavior. Rapid increases in circumferential and axial stresses occurred at lower strains in the older patient group than in the younger. Mean strength was significantly lower in older patients (1.35 +/- 0.37 MPa, n= 14) than younger (2.04 +/- 0.46 MPa, n = 11, age <50 years). These changes in mechanical properties suggest that age may influence the risk of aortic dissection or rupture of dilated ascending aorta.
One-dimensional Viscoelastic Behavior of Fibroblast Populated Collagen Matrices
Bio-artificial tissues are being developed as replacements for damaged biologic tissues. Their mechanical properties are critical for load bearing applications. Current testing protocols for bio-artificial tissues vary widely and often do not consider viscoelasticity. Uniaxial stretch tests were performed on fibroblast populated collagen matrices (FPCMs) to determine the influence of specific test protocols on the mechanical behavior. The peak force, hysteresis and shape of the force-stretch curve are affected by the stretch rate, rest period, stretch amplitude and the number and magnitude of preconditioning cycles.
Cell Orientation Influences the Biaxial Mechanical Properties of Fibroblast Populated Collagen Vessels
Bioartificial tissues, composed of cells in a collagen matrix, can be fabricated with preferred cell orientations to mimic the histologic arrangement of biologic tissues. The influence of preferred cell orientations on the biaxial mechanical behavior of bioartificial tissues is unknown. Characterizing the biaxial mechanical behavior is necessary for better predicting the in vivo behavior of bioartificial tissues. Fibroblast populated collagen vessels (FPCVs) were fabricated with two different cell orientations by controlling the mechanical constraints during incubation. The cell orientation was verified by confocal microscopy and the collagen fiber organization was examined by confocal reflection and scanning electron microscopy (SEM). Pressure-diameter, force-length tests were performed to determine the influence of cell orientation on the biaxial mechanical behavior. FPCVs were more extensible in the direction perpendicular to the preferred cell orientation, than in the direction parallel to the cell orientation. Biaxial tests were also performed in the presence of Cytochalasin D (Cyto D) to minimize the mechanical contribution of the cells. After Cyto D treatment, the FPCVs remained more extensible in the direction perpendicular to the cell orientation, even though a preferred collagen fiber orientation was not observed in the microscopy images.
Effects of Elastin Haploinsufficiency on the Mechanical Behavior of Mouse Arteries
Supravalvular aortic stenosis (SVAS) is associated with decreased elastin and altered arterial mechanics. Mice with a single deletion in the elastin gene (ELN(+/-)) are models for SVAS. Previous studies have shown that elastin haploinsufficiency in these mice causes hypertension, decreased arterial compliance, and changes in arterial wall structure. Despite these differences, ELN(+/-) mice have a normal life span, suggesting that the arteries remodel and adapt to the decreased amount of elastin. To test this hypothesis, we performed in vitro mechanical tests on abdominal aorta, ascending aorta, and left common carotid artery from ELN(+/-) and wild-type (C57BL/6J) mice. We compared the circumferential and longitudinal stress-stretch relationships and residual strains. The circumferential stress-stretch relationship is similar between genotypes and changes <3% with longitudinal stretch at lengths within 10% of the in vivo value. At mean arterial pressure, the circumferential stress in the ascending aorta is higher in ELN(+/-) than in wild type. Although arterial pressures are higher, the increased number of elastic lamellae in ELN(+/-) arteries results in similar tension/lamellae compared with wild type. The longitudinal stress-stretch relationship is similar between genotypes for most arteries. Compared with wild type, the in vivo longitudinal stretch is lower in ELN(+/-) abdominal and carotid arteries and the circumferential residual strain is higher in ELN(+/-) ascending aorta. The increased circumferential residual strain brings the transmural strain distribution in ELN(+/-) ascending aorta close to wild-type values. The mechanical behavior of ELN(+/-) arteries is likely due to the reduced elastin content combined with adaptive remodeling during vascular development.
Elastic Fiber Formation: a Dynamic View of Extracellular Matrix Assembly Using Timer Reporters
To study the dynamics of elastic fiber assembly, mammalian cells were transfected with a cDNA construct encoding bovine tropoelastin in frame with the Timer reporter. Timer is a derivative of the DsRed fluorescent protein that changes from green to red over time and, hence, can be used to distinguish new from old elastin. Using dynamic imaging microscopy, we found that the first step in elastic fiber formation is the appearance of small cell surface-associated elastin globules that increased in size with time (microassembly). The elastin globules are eventually transferred to pre-existing elastic fibers in the extracellular matrix where they coalesce into larger structures (macroassembly). Mechanical forces associated with cell movement help shape the forming, extracellular elastic fiber network. Time-lapse imaging combined with the use of Timer constructs provides unique tools for studying the temporal and spatial aspects of extracellular matrix formation by live cells.
Modeling Cell and Matrix Anisotropy in Fibroblast Populated Collagen Vessels
Microstructurally based models for bio-artificial tissues are needed to predict in vivo mechanical behavior and to validate assumptions for models of biologic tissues. We develop a microstructural model, based on on Zahalak et al. (2000) [Biophys 79(5):2369-2381], to describe matrix and tissue anisotropy observed in recent biaxial tests of fibroblast populated collagen vessels (FPCVs) with different cell orientations (Wagenseil et al. in Ann Biomed Eng 32(5):720-731 2004). The model includes pseudo-elastic cell behavior and pseudo-elastic, non-linear matrix behavior with recruitment of initially buckled collagen fibers. We obtained estimates of collagen matrix parameters from measurements of FPCVs treated with 2x 10(-6) M Cytochalasin D and used these estimates to determine cell parameters in FPCVs activated with 5% fetal calf serum. The estimated stiffness of individual fibroblasts was 41-1,165 kPa. Parameter estimates for both cell and matrix were influenced by the non-linearity of the biaxial test data, making it difficult to obtain unique parameter values for some experiments. Additional microstructural measurements of the collagen matrix may help to more precisely determine the relative contributions of cells and matrix.
Elastin-insufficient Mice Show Normal Cardiovascular Remodeling in 2K1C Hypertension Despite Higher Baseline Pressure and Unique Cardiovascular Architecture
Mice heterozygous for the elastin gene (ELN(+/-)) show unique cardiovascular properties, including increased blood pressure and smaller, thinner arteries with an increased number of lamellar units. Some of these properties are also observed in humans with supravalvular aortic stenosis, a disease caused by functional heterozygosity of the elastin gene. The arterial geometry in ELN(+/-) mice is contrary to the increased thickness that would be expected in an animal demonstrating hypertensive remodeling. To determine whether this is due to a decreased capability for cardiovascular remodeling or to a novel adaptation of the ELN(+/-) cardiovascular system, we increased blood pressure in adult ELN(+/+) and ELN(+/-) mice using the two-kidney, one-clip Goldblatt model of hypertension. Successfully clipped mice have a systolic pressure increase of at least 15 mmHg over sham-operated animals. ELN(+/+) and ELN(+/-)-clipped mice show significant increases over sham-operated mice in cardiac weight, arterial thickness, and arterial cross-sectional area with no changes in lamellar number. There are no significant differences in most mechanical properties with clipping in either genotype. These results indicate that ELN(+/+) and ELN(+/-) hearts and arteries remodel similarly in response to adult induced hypertension. Therefore, the cardiovascular properties of ELN(+/-) mice are likely due to developmental remodeling in response to altered hemodynamics and reduced elastin levels.
New Insights into Elastic Fiber Assembly
Elastic fibers provide recoil to tissues that undergo repeated stretch, such as the large arteries and lung. These large extracellular matrix (ECM) structures contain numerous components, and our understanding of elastic fiber assembly is changing as we learn more about the various molecules associated with the assembly process. The main components of elastic fibers are elastin and microfibrils. Elastin makes up the bulk of the mature fiber and is encoded by a single gene. Microfibrils consist mainly of fibrillin, but also contain or associate with proteins such as microfibril associated glycoproteins (MAGPs), fibulins, and EMILIN-1. Microfibrils were thought to facilitate alignment of elastin monomers prior to cross-linking by lysyl oxidase (LOX). We now know that their role, as well as the overall assembly process, is more complex. Elastic fiber formation involves elaborate spatial and temporal regulation of all of the involved proteins and is difficult to recapitulate in adult tissues. This report summarizes the known interactions between elastin and the microfibrillar proteins and their role in elastic fiber assembly based on in vitro studies and evidence from knockout mice. We also propose a model of elastic fiber assembly based on the current data that incorporates interactions between elastin, LOXs, fibulins and the microfibril, as well as the pivotal role played by cells in structuring the final functional fiber.
Reduced Vessel Elasticity Alters Cardiovascular Structure and Function in Newborn Mice
Elastic blood vessels provide capacitance and pulse-wave dampening, which are critically important in a pulsatile circulatory system. By studying newborn mice with reduced (Eln(+/)(-)) or no (Eln(-)(/)(-)) elastin, we determined the effects of altered vessel elasticity on cardiovascular development and function. Eln(-)(/)(-) mice die within 72 hours of birth but are viable throughout fetal development when dramatic cardiovascular structural and hemodynamic changes occur. Thus, newborn Eln(-)(/)(-) mice provide unique insight into how a closed circulatory system develops when the arteries cannot provide the elastic recoil required for normal heart function. Compared with wild type, the Eln(-)(/)(-) aorta has a smaller unloaded diameter and thicker wall because of smooth muscle cell overproliferation and has greatly reduced compliance. Arteries in Eln(-)(/)(-) mice are also tortuous with stenoses and dilations. Left ventricular pressure is 2-fold higher than wild type, and heart function is impaired. Newborn Eln(+/)(-) mice, in contrast, have normal heart function despite left ventricular pressures 25% higher than wild type. The major vessels have smaller unloaded diameters and longer lengths. The Eln(+/)(-) aorta has additional smooth muscle cell layers that appear in the adventitia at or just before birth. These results show that the major adaptive changes in cardiovascular hemodynamics and in vessel wall structure seen in the adult Eln(+/)(-) mouse are defined in late fetal development. Together, these results show that reduced elastin in mice leads to adaptive remodeling, whereas the complete lack of elastin leads to pathological remodeling and death.
Vascular Extracellular Matrix and Arterial Mechanics
An important factor in the transition from an open to a closed circulatory system was a change in vessel wall structure and composition that enabled the large arteries to store and release energy during the cardiac cycle. The component of the arterial wall in vertebrates that accounts for these properties is the elastic fiber network organized by medial smooth muscle. Beginning with the onset of pulsatile blood flow in the developing aorta, smooth muscle cells in the vessel wall produce a complex extracellular matrix (ECM) that will ultimately define the mechanical properties that are critical for proper function of the adult vascular system. This review discusses the structural ECM proteins in the vertebrate aortic wall and will explore how the choice of ECM components has changed through evolution as the cardiovascular system became more advanced and pulse pressure increased. By correlating vessel mechanics with physiological blood pressure across animal species and in mice with altered vessel compliance, we show that cardiac and vascular development are physiologically coupled, and we provide evidence for a universal elastic modulus that controls the parameters of ECM deposition in vessel wall development. We also discuss mechanical models that can be used to design better tissue-engineered vessels and to test the efficacy of clinical treatments.
Discrete Contributions of Elastic Fiber Components to Arterial Development and Mechanical Compliance
Even though elastin and fibrillin-1 are the major structural components of elastic fibers, mutations in elastin and fibrillin-1 lead to narrowing of large arteries in supravalvular aortic stenosis and dilation of the ascending aorta in Marfan syndrome, respectively. A genetic approach was therefore used here to distinguish the differential contributions of elastin and fibrillin-1 to arterial development and compliance.
The Importance of Elastin to Aortic Development in Mice
Elastin is an essential component of vertebrate arteries that provides elasticity and stores energy during the cardiac cycle. Elastin production in the arterial wall begins midgestation but increases rapidly during the last third of human and mouse development, just as blood pressure and cardiac output increase sharply. The aim of this study is to characterize the structure, hemodynamics, and mechanics of developing arteries with reduced elastin levels and determine the critical time period where elastin is required in the vertebrate cardiovascular system. Mice that lack elastin (Eln(-/-)) or have approximately one-half the normal level (Eln(+/-)) show relatively normal cardiovascular development up to embryonic day (E) 18 as assessed by arterial morphology, left ventricular blood pressure, and cardiac function. Previous work showed that just a few days later, at birth, Eln(-/-) mice die with high blood pressure and tortuous, stenotic arteries. During this period from E18 to birth, Eln(+/-) mice add extra layers of smooth muscle cells to the vessel wall and have a mean blood pressure 25% higher than wild-type animals. These findings demonstrate that elastin is only necessary for normal cardiovascular structure and function in mice starting in the last few days of fetal development. The large increases in blood pressure during this period may push hemodynamic forces over a critical threshold where elastin becomes required for cardiovascular function. Understanding the interplay between elastin amounts and hemodynamic forces in developing vessels will help design treatments for human elastinopathies and optimize protocols for tissue engineering.
A Constrained Mixture Model for Developing Mouse Aorta
Mechanical stresses influence the structure and function of adult and developing blood vessels. When these stresses are perturbed, the vessel wall remodels to return the stresses to homeostatic levels. Constrained mixture models have been used to predict remodeling of adult vessels in response to step changes in blood pressure, axial length and blood flow, but have not yet been applied to developing vessels. Models of developing blood vessels are complicated by continuous and simultaneous changes in the mechanical forces. Understanding developmental growth and remodeling is important for treating human diseases and designing tissue-engineered blood vessels. This study presents a constrained mixture model for postnatal development of mouse aorta with multiple step increases in pressure, length and flow. The baseline model assumes that smooth muscle cells (SMCs) in the vessel wall immediately constrict or dilate the inner radius after a perturbation to maintain the shear stress and then remodel the wall thickness to maintain the circumferential stress. The elastin, collagen and SMCs have homeostatic stretch ratios and passive material constants that do not change with developmental age. The baseline model does not predict previously published experimental data. To approximate the experimental data, it must be assumed that the SMCs dilate a constant amount, regardless of the step change in mechanical forces. It must also be assumed that the homeostatic stretch ratios and passive material constants change with age. With these alterations, the model approximates experimental data on the mechanical properties and dimensions of aorta from 3- to 30-day-old mice.
Decreased Aortic Diameter and Compliance Precedes Blood Pressure Increases in Postnatal Development of Elastin-insufficient Mice
Increased arterial stiffness and blood pressure are characteristic of humans and adult mice with reduced elastin levels caused by aging or genetic disease. Direct associations have been shown between increased arterial stiffness and hypertension in humans, but it is not known whether changes in mechanical properties or increased blood pressure occur first. Using genetically modified mice with elastin haploinsufficiency (Eln(+/-)), we investigated the temporal relationship between arterial mechanical properties and blood pressure throughout postnatal development. Our results show that some mechanical properties are maintained constant regardless of elastin amounts. The peak diameter compliance for both genotypes occurs near the physiologic pressure at each age, which acts to provide maximum pulse dampening. The stress-strain relationships are similar between genotypes and become nonlinear near the systolic pressure for each age, which serves to limit distension under high pressure. Our results also show that some mechanical properties are affected by reduced elastin levels and that these changes occur before measurable changes in blood pressure. Eln(+/-) mice have decreased aortic diameter and compliance in ex vivo tests that are significant by postnatal day 7 and increased blood pressure that is not significant until postnatal day 14. This temporal relationship suggests that targeting large arteries to increase diameter or compliance may be an effective treatment for human hypertension.
Effect of Storage Duration on the Mechanical Behavior of Mouse Carotid Artery
Determining arterial mechanical properties is important for understanding the work done by the heart and how it changes with cardiovascular disease. Ex vivo tests are necessary to apply various loads to the artery and obtain data to model and predict the behavior under any load. Most ex vivo tests are performed within 24 h of dissection, so the tissue is still "alive." For large elastic arteries; however, the passive mechanical behavior is attributed mostly to the very stable proteins, elastin, and collagen. If the testing equipment fails, is in use, or is located at another facility, it would be useful to store the vessels and postpone the tests until the equipment is available. The goal of this study is to determine the effects of storage time on the mechanical behavior of the common carotid artery from adult mice. Each artery was tested after storage for 1-28 days in physiologic saline at 4°C. There were no significant effects of storage time on the arterial diameter or force at each pressure, but there were significant effects on the stretch ratio and stress at each pressure. The significant effects on the stretch ratio and stress were due to decreases in the unloaded dimensions with storage time, when measured from cut arterial rings. When the unloaded dimensions were measured instead from histology sections, there were no significant changes with storage time. We conclude that histology sections yield a more consistent measurement of the unloaded dimensions and that there are no significant changes in the mechanical behavior of mouse carotid artery with storage up to 28 days.
Elastin in Large Artery Stiffness and Hypertension
Large artery stiffness, as measured by pulse wave velocity, is correlated with high blood pressure and may be a causative factor in essential hypertension. The extracellular matrix components, specifically the mix of elastin and collagen in the vessel wall, determine the passive mechanical properties of the large arteries. Elastin is organized into elastic fibers in the wall during arterial development in a complex process that requires spatial and temporal coordination of numerous proteins. The elastic fibers last the lifetime of the organism but are subject to proteolytic degradation and chemical alterations that change their mechanical properties. This review discusses how alterations in the amount, assembly, organization, or chemical properties of the elastic fibers affect arterial stiffness and blood pressure. Strategies for encouraging or reversing alterations to the elastic fibers are addressed. Methods for determining the efficacy of these strategies, by measuring elastin amounts and arterial stiffness, are summarized. Therapies that have a direct effect on arterial stiffness through alterations to the elastic fibers in the wall may be an effective treatment for essential hypertension.
