Diet Alters Age‐related Remodeling of Aortic Extracellular Matrix in Mice Susceptible to Atherosclerosis

Abstract

Atherosclerosis is a chronic inflammatory disease of the arterial wall, constituting a significant source of morbidity and mortality in the United States. In many arteries, remodeling of the collagenous framework occurs as a result of atheromatous plaque development in the intima of the arterial wall, termed atherogenesis. A major challenge in understanding how collagen contributes to the functional properties of the arterial wall is identifying how vascular cells respond to mechanical loading by restructuring the surrounding extracellular matrix. Therefore, the objective of this study is to evaluate collagen architecture to understand the remodeling capabilities of the aorta during aging with or without atherosclerotic lesion development. Groups of 5 apolipoprotein E (apoE) knockout (KO) mice were fed a chow diet for 6 weeks or an additional 6 months. To promote atherosclerotic lesion formation within the aorta, 5 mice were fed a high-fat Western diet (42 kcal% fat) for an additional 6 months. The apoE KO mice are susceptible to atherosclerosis due to a genetic defect in their ability to clear circulating plasma lipoproteins. Aortic diameters were measured in vivo prior to euthanasia using small animal ultrasound (Vevo 770). Following the duration of each diet, mice were euthanized at 6 weeks old or ~7.5 months old. Segments from non-lesioned areas of the thoracic and abdominal aorta were imaged using second harmonic generation (SHG) microscopy. Optical sectioning with SHG microscopy permits visualization of collagen fibers throughout the vessel wall thickness without staining. The images of the collagen fibers were used to determine angle measurements with the circumferential (0°) and axial directions (±90°) serving as the reference points. Image analysis was performed using Continuity 6.4b software. A multiple regression analysis was performed in R to identify variables (mouse age, diet, anatomical location, through-thickness position) having a significant effect on mean fiber angle. Our data indicate that the aortas of 6-week old apoE KO mice have a mean absolute collagen fiber angle of 35.17±1.89° but that fibers become more circumferentially aligned at 12.52° after 6 months of Western diet feeding. In contrast, collagen fiber average absolute angle undergoes a significant increase in apoE KO mice fed a chow diet for 6 months, with fibers becoming more axially aligned, at 61.61°. Six-month-old mice on Western diet demonstrate increased gain in body weight compared to 6-month-old mice on chow diet (31.05±4.21g vs 24.53±2.99g p<0.05), which further supports differences in these mice independent of aging. Body weight for 6-week-old mice was 16.45±2.55g. However, aortic diameters were not significantly different between mice on chow diet for 6 months vs. those on Western diet. We conclude that the aorta, while showing an equivalent increase in collagen content during aging, displays distinct remodeling responses in the presence of atherogenic stimuli, even in non-lesioned areas. Acknowledgements: Special thanks to Dr. Bruce Gao and Zhonghai Wang at Clemson University for use of the SHG microscope. Support or Funding Information This work was supported by National Science Foundation grants EPS-0903795 and CMMI-1200358, and National Institute of Health grants P30 GM103336-01A1 and R01 CA154731. Average collagen fiber angle diverges with aging in apoE KO mice on chow diet vs. Western diet. Compared to 6-week old apoE KO mice, multiple regression analysis showed a significant difference in average absolute fiber angle in response to age and high-fat diet. On average, absolute fiber angle was increased by 26.44±2.67° in older mice on same diet and reduced 22.35±2.71° in mice fed a Western diet for 6 months. Collagen content (% area) for each of the three treatment groups obtained from Picrosirius Red staining. Collagen content increases significantly with age from 6 weeks to 7.5 months (P<0.05), but is not altered by Western diet. CD = chow diet; WD = Western diet