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.

Biomechanical Regulation of Mesenchymal Stem Cells for Cardiovascular Tissue Engineering.

Mesenchymal stem cells (MSCs) are an appealing potential therapy for vascular diseases; however, many challenges remain in their clinical translation. While the use of biochemical, pharmacological, and substrate-mediated treatments to condition MSCs has been subjected to intense investigation, there has been far less exploration of using these treatments in combination with applied mechanical force for conditioning MSCs toward vascular phenotypes. This review summarizes the current understanding of the use of applied mechanical forces to differentiate MSCs into vascular cells and enhance their therapeutic potential for cardiovascular disease. First recent work on the use of material-based mechanical cues for differentiation of MSCs into vascular and cardiovascular phenotypes is examined. Then a summary of the studies using mechanical stretch or shear stress in combination with biochemical treatments to enhance vascular phenotypes in MSCs is presented.

Critical buckling pressure in mouse carotid arteries with altered elastic fibers.

Arteries can buckle axially under applied critical buckling pressure due to a mechanical instability. Buckling can cause arterial tortuosity leading to flow irregularities and stroke. Genetic mutations in elastic fiber proteins are associated with arterial tortuosity in humans and mice, and may be the result of alterations in critical buckling pressure. Hence, the objective of this study is to investigate how genetic defects in elastic fibers affect buckling pressure. We use mouse models of human disease with reduced amounts of elastin (Eln+/-) and with defects in elastic fiber assembly due to the absence of fibulin-5 (Fbln5-/-). We find that Eln+/- arteries have reduced buckling pressure compared to their wild-type controls. Fbln5-/- arteries have similar buckling pressure to wild-type at low axial stretch, but increased buckling pressure at high stretch. We fit material parameters to mechanical test data for Eln+/-, Fbln5-/- and wild-type arteries using Fung and four-fiber strain energy functions. Fitted parameters are used to predict theoretical buckling pressure based on equilibrium of an inflated, buckled, thick-walled cylinder. In general, the theoretical predictions underestimate the buckling pressure at low axial stretch and overestimate the buckling pressure at high stretch. The theoretical predictions with both models replicate the increased buckling pressure at high stretch for Fbln5-/- arteries, but the four-fiber model predictions best match the experimental trends in buckling pressure changes with axial stretch. This study provides experimental and theoretical methods for further investigating the influence of genetic mutations in elastic fibers on buckling behavior and the development of arterial tortuosity.

Mechanical factors direct mouse aortic remodelling during early maturation.

Numerous diseases have been linked to genetic mutations that lead to reduced amounts or disorganization of arterial elastic fibres. Previous work has shown that mice with reduced amounts of elastin (Eln+/-) are able to live a normal lifespan through cardiovascular adaptations, including changes in haemodynamic stresses, arterial geometry and arterial wall mechanics. It is not known if the timeline and presence of these adaptations are consistent in other mouse models of elastic fibre disease, such as those caused by the absence of fibulin-5 expression (Fbln5-/-). Adult Fbln5-/- mice have disorganized elastic fibres, decreased arterial compliance and high blood pressure. We examined mechanical behaviour of the aorta in Fbln5-/- mice through early maturation when the elastic fibres are being assembled. We found that the physiologic circumferential stretch, stress and modulus of Fbln5-/- aorta are maintained near wild-type levels. Constitutive modelling suggests that elastin contributions to the total stress are decreased, whereas collagen contributions are increased. Understanding how collagen fibre structure and mechanics compensate for defective elastic fibres to meet the mechanical requirements of the maturing aorta may help to better understand arterial remodelling in human elastinopathies.

Measuring, reversing, and modeling the mechanical changes due to the absence of Fibulin-4 in mouse arteries.

Mice with a smooth muscle cell (SMC)-specific deletion of Fibulin-4 (SMKO) show decreased expression of SMC contractile genes, decreased circumferential compliance, and develop aneurysms in the ascending aorta. Neonatal administration of drugs that inhibit the angiotensin II pathway encourages the expression of contractile genes and prevents aneurysm development, but does not increase compliance in SMKO aorta. We hypothesized that multidimensional mechanical changes in the aorta and/or other elastic arteries may contribute to aneurysm pathophysiology. We found that the SMKO ascending aorta and carotid artery showed mechanical changes in the axial direction. These changes were not reversed by angiotensin II inhibitors, hence reversing the axial changes is not required for aneurysm prevention. Mechanical changes in the circumferential direction were specific to the ascending aorta; therefore, mechanical changes in the carotid do not contribute to aortic aneurysm development. We also hypothesized that a published model of postnatal aortic growth and remodeling could be used to investigate mechanisms behind the changes in SMKO aorta and aneurysm development over time. Dimensions and mechanical behavior of adult SMKO aorta were reproduced by the model after modifying the initial component material constants and the aortic dilation with each postnatal time step. The model links biological observations to specific mechanical responses in aneurysm development and treatment.

Fibulin-5 null mice with decreased arterial compliance maintain normal systolic left ventricular function, but not diastolic function during maturation.

Abstract The large arteries serve as compliant vessels that store energy during systole and return it during diastole. This function is made possible by the elastic fibers in the arterial wall that are assembled during late embryonic and early postnatal development from various proteins, including fibulin-5. Mice and humans with insufficient amounts of fibulin-5 have reduced arterial compliance as adults. Reduced compliance of the large arteries is correlated with hypertension, reduced cardiac function, and an increased risk of death from cardiac and cardiovascular disease. The goal of this study was to quantify arterial compliance, blood pressure, and left ventricular (LV) function from early postnatal development to young adulthood in fibulin-5 null (Fbln5-/-) mice to determine the effects of reduced arterial compliance during this critical period of elastic fiber assembly. We find that ascending aorta compliance is reduced as early as postnatal day (P) 7 and carotid artery compliance is reduced by P21 in Fbln5-/- mice. We did not find significant increases in systolic blood pressure by P60, but pulse pressures are increased by P21 in Fbln5-/- mice. LV systolic function, as measured by ejection fraction and fractional shortening, is unaffected in Fbln5-/- mice. However, LV diastolic function, as measured by tissue Doppler imaging, is compromised at all ages in Fbln5-/- mice. We propose that Fbln5-/- mice represent a suitable model for further studies to determine mechanistic relationships between arterial compliance and LV diastolic function.

Angiotensin-converting enzyme-induced activation of local angiotensin signaling is required for ascending aortic aneurysms in fibulin-4-deficient mice.

Aortic aneurysms are life-threatening and often associated with defects in connective tissues and mutations in smooth muscle cell (SMC) contractile proteins. Despite recent advances in understanding altered signaling in aneurysms of Marfan syndrome, the underlying mechanisms and options for pharmacological treatment for other forms of aneurysms are still under investigation. We previously showed in mice that deficiency in the fibulin-4 gene in vascular SMCs (Fbln4(SMKO)) leads to loss of the SMC contractile phenotype, hyperproliferation, and ascending aortic aneurysms. We report that abnormal up-regulation of angiotensin-converting enzyme (ACE) in SMCs and subsequent activation of angiotensin II (AngII) signaling are involved in the onset of aortic aneurysms in Fbln4(SMKO) mice. In this model, aneurysm formation was completely prevented by inhibition of the AngII pathway with losartan or captopril within a narrow therapeutic window during the first month of life, even though the altered mechanical properties of blood vessel walls were not reversed by the pharmacological treatment. The therapeutic effects of losartan in Fbln4(SMKO) mice do not require the AngII receptor type 2 (Agtr2) but likely require both type 1a (Agtr1a) and 1b (Agtr1b) receptors. The results indicate that fibulin-4 is a vascular matrix component required for regulation of local angiotensin signaling and development and maintenance of the SMC phenotype.

Echocardiographic Characterization of Postnatal Development in Mice with Reduced Arterial Elasticity.

PURPOSE: Decreased expression of elastin results in smaller, less compliant arteries and high blood pressure. In mice, these differences become more significant with postnatal development. It is known that arterial size and compliance directly affect cardiac function, but the temporal changes in cardiac function have not been investigated in elastin insufficient mice. The aim of this study is to correlate changes in arterial size and compliance with cardiac function in wildtype (WT) and elastin haploinsufficient (Eln+/- ) mice from birth to adulthood. METHODS: Ultrasound scans were performed at the ages of 3, 7, 14, 21, 30, 60, and 90 days on male and female WT and Eln+/- mice. 2-D ultrasound and pulse wave Doppler images were used to measure the dimensions and function of the left ventricle (LV), ascending aorta and carotid arteries. RESULTS: Eln+/- arteries are smaller and less compliant at most ages, with significant differences from WT as early as 3 days old. Surprisingly, there are no correlations (R2 < 0.2) between arterial size and compliance with measures of LV hypertrophy or systolic function. There are weak correlations (0.2 < R2 < 0.5) between arterial size and compliance with measures of LV diastolic function. CONCLUSIONS: Eln+/- mice have similar cardiac function to WT throughout postnatal development, demonstrating the remarkable ability of the developing cardiovascular system to adapt to mechanical and hemodynamic changes. Correlations between arterial size and compliance with diastolic function show that these measures may be useful indicators of early diastolic dysfunction.

Measuring left ventricular pressure in late embryonic and neonatal mice.

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable around embryonic day (E) 8.5(1). Systolic left ventricular (LV) pressure is 2 mmHg at E9.5 and 11 mmHg at E14.5(2). At these mid-embryonic stages, the LV is clearly visible through the chest wall for invasive pressure measurements because the ribs and skin are not fully developed. Between E14.5 and birth (approximately E21) imaging methods must be used to view the LV. After birth, mean arterial pressure increases from 30 - 70 mmHg from postnatal day (P) 2 - 35(3). Beyond P20, arterial pressure can be measured with solid-state catheters (i.e. Millar or Scisense). Before P20, these catheters are too big for developing mouse arteries and arterial pressure must be measured with custom pulled plastic catheters attached to fluid-filled pressure transducers(3) or glass micropipettes attached to servo null pressure transducers(4). Our recent work has shown that the greatest increase in blood pressure occurs during the late embryonic to early postnatal period in mice(5-7). This large increase in blood pressure may influence smooth muscle cell (SMC) phenotype in developing arteries and trigger important mechanotransduction events. In human disease, where the mechanical properties of developing arteries are compromised by defects in extracellular matrix proteins (i.e. Marfan's Syndrome(8) and Supravalvular Aortic Stenosis(9)) the rapid changes in blood pressure during this period may contribute to disease phenotype and severity through alterations in mechanotransduction signals. Therefore, it is important to be able to measure blood pressure changes during late embryonic and neonatal periods in mouse models of human disease. We describe a method for measuring LV pressure in late embryonic (E18) and early postnatal (P1 - 20) mice. A needle attached to a fluid-filled pressure transducer is inserted into the LV under ultrasound guidance. Care is taken to maintain normal cardiac function during the experimental protocol, especially for the embryonic mice. Representative data are presented and limitations of the protocol are discussed.

Mechanical testing of mouse carotid arteries: from newborn to adult.

The large conducting arteries in vertebrates are composed of a specialized extracellular matrix designed to provide pulse dampening and reduce the work performed by the heart. The mix of matrix proteins determines the passive mechanical properties of the arterial wall(1). When the matrix proteins are altered in development, aging, disease or injury, the arterial wall remodels, changing the mechanical properties and leading to subsequent cardiac adaptation(2). In normal development, the remodeling leads to a functional cardiac and cardiovascular system optimized for the needs of the adult organism. In disease, the remodeling often leads to a negative feedback cycle that can cause cardiac failure and death. By quantifying passive arterial mechanical properties in development and disease, we can begin to understand the normal remodeling process to recreate it in tissue engineering and the pathological remodeling process to test disease treatments. Mice are useful models for studying passive arterial mechanics in development and disease. They have a relatively short lifespan (mature adults by 3 months and aged adults by 2 years), so developmental(3) and aging studies(4) can be carried out over a limited time course. The advances in mouse genetics provide numerous genotypes and phenotypes to study changes in arterial mechanics with disease progression(5) and disease treatment(6). Mice can also be manipulated experimentally to study the effects of changes in hemodynamic parameters on the arterial remodeling process(7). One drawback of the mouse model, especially for examining young ages, is the size of the arteries. We describe a method for passive mechanical testing of carotid arteries from mice aged 3 days to adult (approximately 90 days). We adapt a commercial myograph system to mount the arteries and perform multiple pressure or axial stretch protocols on each specimen. We discuss suitable protocols for each age, the necessary measurements and provide example data. We also include data analysis strategies for rigorous mechanical characterization of the arteries.