Jessica E. Wagenseil

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.

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.

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. J. Cell. Physiol. 207: 87–96, 2006.

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.

Discrete Contributions of Elastic Fiber Components to Arterial Development and Mechanical Compliance

Objective—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 supravascular 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. Methods and Results—Key parameters of cardiovascular function were compared among adult mice haploinsufficient for elastin (Eln+/−), fibrillin-1 (Fbn1+/−), or both proteins (dHet). Physiological and morphological comparisons correlate elastin haploinsufficiency with increased blood pressure and vessel length and tortuosity in dHet mice, and fibrillin-1 haploinsufficiency with increased aortic diameter in the same mutant animals. Mechanical tests confirm that elastin and fibrillin-1 impart elastic recoil and tensile strength to the aortic wall, respectively. Additional ex vivo analyses demonstrate additive and overlapping contributions of elastin and fibrillin-1 to the material properties of vascular tissues. Lastly, light and electron microscopy evidence implicates fibrillin-1 in the hypertension-promoted remodeling of the elastin-deficient aorta. Conclusions—These results demonstrate that elastin and fibrillin-1 have both differential and complementary roles in arterial wall formation and function, and advance our knowledge of the structural determinants of vascular physiology and disease.

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.

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.

Characterization of t1 relaxation and blood‐myocardial contrast enhancement of NC100150 injection in cardiac MRI

A new ultrasmall superparamagnetic iron oxide (Clariscan; NC100150 Injection) was studied in domestic farm pigs. The T1 effects were characterized for blood and myocardium and the blood‐myocardial contrast was measured in T1‐weighted cine images. The contrast‐to‐noise ratio (CNR) and signal‐to‐noise ratio (SNR) were measured at baseline and contrast doses of 1 and 5 mg Fe/kg body weight (bw) at end diastole and late systole. The T1 values for blood and myocardium were reduced by 97 and 43%, respectively, from baseline to 5 mg Fe/kg bw. The CNR was significantly improved with contrast at end diastole and late systole. The maximum improvement shown was 202% at 5 mg Fe/kg bw in late systole. The percent SNR enhancement was significantly higher in blood than myocardium at late systole. NC100150 Injection is an effective T1 shortening agent and can be used to improve blood‐myocardial contrast in cine images of the heart. J. Magn. Reson. Imaging 1999;10:784–789.

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.

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.