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The endothelium, composed of a single layer of endothelial cells, is the innermost lining of vessels, acting as the interface between blood and the arterial wall. “Endothelial dysfunction” is defined as reduction or loss of bioavailability of endothelial-derived nitric oxide (NO), a condition that precedes or accompanies several cardiovascular pathologies associated with aging, such as atherosclerosis. [1] [2] NO plays an important role in regulating vascular tone and maintaining vascular homeostasis as a vasodilator. Thus, we hypothesize that decreased NO production may induce collagen fiber reorientation and increased collagen production, to shift load from smooth muscle cells to the extracellular matrix, eventually leading to vascular remodeling. The aim of this project is to study the impact of NO deficiency on hemodynamic parameters, collagen content, and collagen fiber orientation during age-related vascular remodeling using a mouse model. 

A novel method utilizing colloidal gold particles to create speckle patterns on mouse aortas for digital image correlation (DIC) studies is presented. The colloidal gold particles form random, stable, high contrast, variably sized speckles suitable for DIC analysis and can be used to measure local strains during mechanical failure testing. 

Vascular cells restructure extracellular matrix in response to aging or changes in mechanical loading. Here, we characterized collagen architecture during age-related aortic remodeling in atherosclerosis-prone mice. We hypothesized that changes in collagen fiber orientation reflect an altered balance between passive and active forces acting on the arterial wall. We examined two factors that can alter this balance, endothelial dysfunction and reduced smooth muscle cell (SMC) contractility. Collagen fiber organization was visualized by second-harmonic generation microscopy in aortic adventitia of apolipoprotein E (apoE) knockout (KO) mice at 6 wk and 6 mo of age on a chow diet and at 7.5 mo of age on a Western diet (WD), using image analysis to yield mean fiber orientation. Adventitial collagen fibers became significantly more longitudinally oriented with aging in apoE knockout mice on chow diet. Conversely, fibers became more circumferentially oriented with aging in mice on WD. Total collagen content increased significantly with age in mice fed WD. We compared expression of endothelial nitric oxide synthase and acetylcholine-mediated nitric oxide release but found no evidence of endothelial dysfunction in older mice. Time-averaged volumetric blood flow in all groups showed no significant changes. Wire myography of aortic rings revealed decreases in active stress generation with age that were significantly exacerbated in WD mice. We conclude that the aorta displays a distinct remodeling response to atherogenic stimuli, indicated by altered collagen organization. Collagen reorganization can occur in the absence of altered hemodynamics and may represent an adaptive response to reduced active stress generation by vascular SMCs. NEW & NOTEWORTHY The following major observations were made in this study: 1) aortic adventitial collagen fibers become more longitudinally oriented with aging in apolipoprotein E knockout mice fed a chow diet; 2) conversely, adventitial collagen fibers become more circumferentially oriented with aging in apoE knockout mice fed a high-fat diet; 3) adventitial collagen content increases significantly with age in mice on a high-fat diet; 4) these alterations in collagen organization occur largely in the absence of hemodynamic changes; and 5) circumferential reorientation of collagen is associated with decreased active force generation (contractility) in aged mice on a high-fat diet. 

Digital image correlation (DIC) is a non-destructive and non-contact optical technique to measure deformation and strain of materials. The method is based on optically tracking the displacements of a speckle pattern created on the material surface. In the case of soft tissues such as mouse aorta, there are several advantages to using DIC since it can provide local, rather than global, deformations and it is suitable for large strain measurements, typical of soft tissues taken to failure [1] [2]. For the optimal use of DIC, several requirements should be met for speckle patterning: 1) randomness, 2) high contrast, 3) appropriate size of speckle in the field of view (3-5 pixels), and 4) firm attachment of speckle to specimen during deformation. In previous DIC studies of soft tissues, the methods employed to create a speckle pattern include the use of an airbrush to spray dye or paint on the specimen, or coating the sample with toner powder. However, biological samples must be partially dehydrated before applying paint which may affect the mechanical properties of the specimen, and toner powder is too hydrophobic to adhere well on specimens when submerged in aqueous solution during mechanical testing. In addition, it is difficult to evenly distribute paint or toner powder on the surface of a hydrated biological specimen [2]. Therefore, a novel method utilizing colloidal gold particles to create a speckle pattern on mouse aorta is proposed in this work.