Compromised homeostasis in aged carotid arteries revealed by microstructural studies of elastic lamellae.

Healthy arteries are continuously subjected to diverse mechanical stimuli and adapt in order to maintain a mechanical homeostasis which is characterized by a uniform distribution of wall stresses. However, aging may compromise the homeostatic microenvironment within arteries. Structural heterogeneity has been suggested as a potential microstructural mechanism that could lead to homogeneous stress distribution across the arterial wall. Our previous study on the unfolding and stretching of the elastic lamellae revealed the underlying microstructural mechanism for equalizing the circumferential stresses through wall; inner elastic layers are wavier and unfold more than the outer layers which helps to evenly distribute lamellar stretching (Yu et al., 2018). In this study, we investigated the effect of aging on lamellar deformation and its implications for tissue homeostasis. Common carotid arteries from aged mice were imaged under a multi-photon microscope while subjected to biaxial extension and inflation at five different pressures ranging from 0 up to 120 mmHg. Lamellar unfolding during pressurization was then determined from the reconstructed cross-sectional images of elastic lamellae. Tissue-level circumferential stretch was combined with the lamellar unfolding to calculate lamellar stretching. Our results revealed that the straightness gradient of aged elastic lamellae is similar to the young ones. However, during pressurization, the inner elastic lamella of the aged mice unfolded significantly more than the inner layer in young arteries. An important finding of our study is the uneven increase in inter-lamellar space which contributed to a nonuniform stretching of the elastic lamellae of aged mice arteries, elevated stress gradient, and a shifting of the load-bearing component to adventitia. Our results shed light into the complex microstructural mechanisms that take place in aging and adversely affect arterial mechanical behavior and homeostasis.

A discrete fiber network finite element model of arterial elastin network considering inter-fiber crosslinking property and density.

Inter-fiber crosslinks within the extracellular matrix (ECM) play important roles in determining the mechanical properties of the fibrous network. Discrete fiber network (DFN) models have been used to study fibrous biological material, however the contribution of inter-fiber crosslinks to the mechanics of the ECM network is not well understood. In this study, a DFN model of arterial elastin network was developed based on measured structural features to study the contribution of inter-fiber crosslinking properties and density to the mechanics and fiber kinematics of the network. The DFN was generated by randomly placing line segments into a given domain following a fiber orientation distribution function obtained from multiphoton microscopy until a desired fiber areal fraction was reached. Intersections between the line segments were treated as crosslinks. The generated DFN model was then incorporated into an ABAQUS finite element model to simulate the network under equi- and nonequi-biaxial deformation. The inter-fiber crosslinks were modeled using connector elements with either zero (pin joint) or infinite (weld joint) rotational stiffness. Furthermore, inter-fiber crosslinking density was systematically reduced and its effect on both network- and fiber-level mechanics was studied. The DFN model showed good fitting and predicting capabilities of the stress-strain behavior of the elastin network. While the pin and weld joints do not seem to have noticeable effect on the network stress-strain behavior, the crosslinking properties can affect the local fiber mechanics and kinematics. Overall, our study suggests that inter-fiber crosslinking properties are important to the multiscale mechanics and fiber kinematics of the ECM network.

Biomechanical Properties of Mouse Carotid Arteries With Diet-Induced Metabolic Syndrome and Aging.

Metabolic syndrome increases the risk of cardiovascular diseases. Arteries gradually stiffen with aging; however, it can be worsened by the presence of conditions associated with metabolic syndrome. In this study, we investigated the combined effects of diet-induced metabolic syndrome and aging on the biomechanical properties of mouse common carotid arteries (CCA). Male mice at 2 months of age were fed a normal or a high fat and high sucrose (HFHS) diet for 2 (young group), 8 (adult group) and 18-20 (old group) months. CCAs were excised and subjected to in vitro biaxial inflation-extension tests and the Cauchy stress-stretch relationships were determined in both the circumferential and longitudinal directions. The elastic energy storage of CCAs was obtained using a four-fiber family constitutive model, while the material stiffness in the circumferential and longitudinal directions was computed. Our study showed that aging is a dominant factor affecting arterial remodeling in the adult and old mice, to a similar extent, with stiffening manifested with a significantly reduced capability of energy storage by ∼50% (p < 0.05) and decreases in material stiffness and stress (p < 0.05), regardless of diet. On the other hand, high fat high sucrose diet resulted in an accelerated arterial remodeling in the young group at pre-diabetic stage by affecting the circumferential material stiffness and stress (p < 0.05), which was eventually overshadowed by aging progression. These findings have important implications on the effects of metabolic syndrome on elastic arteries in the younger populations.

Mechanical and structural contributions of elastin and collagen fibers to interlamellar bonding in the arterial wall.

The artery relies on interlamellar structural components, mainly elastin and collagen fibers, for maintaining its integrity and resisting dissection propagation. In this study, the contribution of arterial elastin and collagen fibers to interlamellar bonding was studied through mechanical testing, multiphoton imaging and finite element modeling. Steady-state peeling experiments were performed on porcine aortic media and the purified elastin network in the circumferential (Circ) and longitudinal (Long) directions. The peeling force and energy release rate associated with mode-I failure are much higher for aortic media than for the elastin network. Also, longitudinal peeling exhibits a higher energy release rate and strength than circumferential peeling for both the aortic media and elastin. Multiphoton imaging shows the recruitment of both elastin and collagen fibers within the interlamellar space and points to in-plane anisotropy of fiber distributions as a potential mechanism for the direction-dependent phenomena of peeling tests. Three-dimensional finite element models based on cohesive zone model (CZM) of fracture were created to simulate the peeling tests with the interlamellar energy release rate and separation distance at damage initiation obtained directly from peeling test. Our experimental results show that the separation distance at damage initiation is 80 µm for aortic media and 40 µm for elastin. The damage initiation stress was estimated from the model for aortic media (Circ: 60 kPa; Long: 95 kPa) and elastin (Circ: 9 kPa; Long: 14 kPa). The interlamellar separation distance at complete failure was estimated to be 3 - 4 mm for both media and elastin. Furthermore, elastin and collagen fibers both play an important role in bonding of the arterial wall, while collagen has a higher contribution than elastin to interlamellar stiffness, strength and toughness. These results on microstructural interlamellar failure shed light on the pathological development and progression of aortic dissection.

Avalanches and power law behavior in aortic dissection propagation.

Aortic dissection is a devastating cardiovascular disease known for its rapid propagation and high morbidity and mortality. The mechanisms underlying the propagation of aortic dissection are not well understood. Our study reports the discovery of avalanche-like failure of the aorta during dissection propagation that results from the local buildup of strain energy followed by a cascade failure of inhomogeneously distributed interlamellar collagen fibers. An innovative computational model was developed that successfully describes the failure mechanics of dissection propagation. Our study provides the first quantitative agreement between experiment and model prediction of the dissection propagation within the complex extracellular matrix (ECM). Our results may lead to the possibility of predicting such catastrophic events based on microscopic features of the ECM.

MMP12 Deletion Preferentially Attenuates Axial Stiffening of Aging Arteries.

Arterial stiffening is a hallmark of aging, but how aging affects the arterial response to pressure is still not completely understood, especially with regard to specific matrix metalloproteinases (MMPs). Here, we performed biaxial inflation-extension tests on C57BL/6 mice to study the effects of age and MMP12, a major arterial elastase, on arterial biomechanics. Aging from 2 to 24 months leads to both circumferential and axial stiffening with stretch, and these changes are associated with an increased wall thickness, a decreased inner radius-wall thickness ratio, and a decreased in vivo axial stretch. Analysis of in vivo stretch and stress-stretch curves with arteries from age- and sex-matched wild-type (WT) and MMP12-null arteries demonstrates that MMP12 deletion attenuates age-dependent arterial stiffening, mostly in the axial direction. MMP12 deletion also prevents the aging-associated decrease in the in vivo stretch and, in general, leads to an axial mechanics phenotype characteristic of much younger mice. Circumferential arterial mechanics were much less affected by deletion of MMP12. We conclude that the induction of MMP12 during aging preferentially promotes axial arterial stiffening.

Micromechanics of elastic lamellae: unravelling the role of structural inhomogeneity in multi-scale arterial mechanics.

Microstructural deformation of elastic lamellae plays important roles in maintaining arterial tissue homeostasis and regulating vascular smooth muscle cell fate. Our study unravels the underlying microstructural origin that enables elastic lamellar layers to evenly distribute the stresses through the arterial wall caused by intraluminal distending pressure, a fundamental requirement for tissue and cellular function. A new experimental approach was developed to quantify the spatial organization and unfolding of elastic lamellar layers under pressurization in mouse carotid arteries by coupling physiological extension-inflation and multiphoton imaging. Tissue-level circumferential stretch was obtained from analysis of the deformation of a thick-walled cylinder. Our results show that the unfolding and extension of lamellar layers contribute simultaneously to tissue-level deformation. The inner lamellar layers are wavier and unfold more than the outer layers. This waviness gradient compensates the larger tissue circumferential stretch experienced at the inner surface, thus equalizing lamellar layer extension through the arterial wall. Discoveries from this study reveal the importance of structural inhomogeneity in maintaining tissue homeostasis through the arterial wall, and may have profound implications on vascular remodelling in aging and diseases, as well as in tissue engineering of functional blood vessels.

Transmural variation in elastin fiber orientation distribution in the arterial wall.

The complex three-dimensional elastin network is a major load-bearing extracellular matrix (ECM) component of an artery. Despite the reported anisotropic behavior of arterial elastin network, it is usually treated as an isotropic material in constitutive models. Our recent multiphoton microscopy study reported a relatively uniform elastin fiber orientation distribution in porcine thoracic aorta when imaging from the intima side (Chow et al., 2014). However it is questionable whether the fiber orientation distribution obtained from a small depth is representative of the elastin network structure in the arterial wall, especially when developing structure-based constitutive models. To date, the structural basis for the anisotropic mechanical behavior of elastin is still not fully understood. In this study, we examined the transmural variation in elastin fiber orientation distribution in porcine thoracic aorta and its association with elastin anisotropy. Using multi-photon microscopy, we observed that the elastin fibers orientation changes from a relatively uniform distribution in regions close to the luminal surface to a more circumferential distribution in regions that dominate the media, then to a longitudinal distribution in regions close to the outer media. Planar biaxial tensile test was performed to characterize the anisotropic behavior of elastin network. A new structure-based constitutive model of elastin network was developed to incorporate the transmural variation in fiber orientation distribution. The new model well captures the anisotropic mechanical behavior of elastin network under both equi- and nonequi-biaxial loading and showed improvements in both fitting and predicting capabilities when compared to a model that only considers the fiber orientation distribution from the intima side. We submit that the transmural variation in fiber orientation distribution is important in characterizing the anisotropic mechanical behavior of elastin network and should be considered in constitutive modeling of an artery.

Vascular Smooth Muscle Sirtuin-1 Protects Against Aortic Dissection During Angiotensin II-Induced Hypertension.

BACKGROUND: Sirtuin-1 (SirT1), a nicotinamide adenine dinucleotide(+)-dependent deacetylase, is a key enzyme in the cellular response to metabolic, inflammatory, and oxidative stresses; however, the role of endogenous SirT1 in the vasculature has not been fully elucidated. Our goal was to evaluate the role of vascular smooth muscle SirT1 in the physiological response of the aortic wall to angiotensin II, a potent hypertrophic, oxidant, and inflammatory stimulus. METHODS AND RESULTS: Mice lacking SirT1 in vascular smooth muscle (ie, smooth muscle SirT1 knockout) had drastically high mortality (70%) caused by aortic dissection after angiotensin II infusion (1 mg/kg per day) but not after an equipotent dose of norepinephrine, despite comparable blood pressure increases. Smooth muscle SirT1 knockout mice did not show any abnormal aortic morphology or blood pressure compared with wild-type littermates. Nonetheless, in response to angiotensin II, aortas from smooth muscle SirT1 knockout mice had severely disorganized elastic lamellae with frequent elastin breaks, increased oxidant production, and aortic stiffness compared with angiotensin II-treated wild-type mice. Matrix metalloproteinase expression and activity were increased in the aortas of angiotensin II-treated smooth muscle SirT1 knockout mice and were prevented in mice overexpressing SirT1 in vascular smooth muscle or with use of the oxidant scavenger tempol. CONCLUSIONS: Endogenous SirT1 in aortic smooth muscle is required to maintain the structural integrity of the aortic wall in response to oxidant and inflammatory stimuli, at least in part, by suppressing oxidant-induced matrix metalloproteinase activity. SirT1 activators could potentially be a novel therapeutic approach to prevent aortic dissection and rupture in patients at risk, such as those with hypertension or genetic disorders, such as Marfan's syndrome.