The pro-atherogenic response to disturbed blood flow is increased by a western diet, but not by old age

Figures

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  Figure 1. Time course for changes in oscillatory blood flow index and wall thickness after partial carotid ligation surgery in young mice and lack of differences between age and diet groups for the oscillatory blood flow response to partial carotid ligation. (A) Oscillatory blood flow index and (B) wall thickness measured by ultrasound in the left (ligated) and right (un-ligated) carotid arteries at pre-surgery and 1, 2, and 3 weeks post-surgery in young, normal chow fed B6D2F1 mice undergoing partial carotid ligation or sham surgery. “Un-ligated Right” is the contralateral (control) to the artery that underwent partial carotid ligation (“Ligated Left”). “Sham Right” is the contralateral (control) to the artery that underwent sham surgery (“Sham Left”). n = 4–5/group. *p < 0.05 vs. pre-surgery ligated left. †p < 0.05 vs. pre-surgery sham left. (C) Oscillatory blood flow index in the un-ligated and ligated carotid arteries after partial carotid ligation surgery in young and old normal chow and western diet fed B6D2F1 mice. n = 6–11/group. *p < 0.05 RM-ANOVA un-ligated vs. ligated. Values are mean ± SEM.

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  Figure 2. Western diet, but not old age, increases pro-atherogenic remodeling in response to partial carotid ligation. (A) Intima media thickness and (B) neointima area of the un-ligated and ligated carotid artery at 3-weeks after partial carotid ligation surgery in young and old, normal chow and western diet fed B6D2F1 mice. Neointima is calculated as the percent area within the internal elastic lamina that is composed of cells. Representative images are shown below. Elastic laminae stain dark brown-black with Verhoff-Van Gieson stain; scale bar = 100 µm. n = 7–11/group. *p < 0.05 RM-ANOVA un-ligated vs. ligated. †p < 0.05 ANOVA main effect normal chow vs. western diet, ‡p < 0.05 ANOVA interaction of ligation × diet.

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  Figure 3. Arterial inflammation is greater with western diet, but not old age, in response to partial carotid ligation. In the young normal chow ligated carotid artery (A), infiltration of mononuclear inflammatory cells (blue arrow) in the adventitia is minimal and inflammation is absent in the neointima, within the internal elastic lamina (black arrowheads). In the western diet fed young ligated carotid artery (B), inflammatory cells (blue arrow) are dense within the adventitia, and neutrophils and mononuclear inflammatory cells (black arrow) also extend into the neointima. (C) The neointima is composed of vascular smooth muscle cells and extracellular matrix within the internal elastic lamina (black arrowheads). Macrophage content measured by the percent of F4/80 positive cells in the arterial wall in the ligated and un-ligated carotid arteries at 3-weeks after partial carotid ligation surgery in young and old, normal chow and western diet fed B6D2F1 mice n = 4–5/group. (A,B) are hematoxylin and eosin stain. Ad, adventitia; NI, neointima; TM, tunica muscularis; L, lumen. Below (C) are representative DAB stained F4/80, with hematoxylin stained nuclei. *p < 0.05 RM-ANOVA un-ligated vs.…

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  Figure 4. Arterial nitrotyrosine content is greater with old age, but not with western diet or partial carotid ligation. Nitrotyrosine content measured by immunofluorescence in the arterial wall in ligated and un-ligated carotid arteries 3-weeks after partial carotid ligation surgery in young and old, normal chow and western diet fed B6D2F1 mice. Representative images are below with nitrotyrosine in red, DAPI nuclei in blue, and autofluorescence of the elastic lamina in green. n = 7–9/group. Values are normalized to normal chow fed young left artery. *p < 0.05 RM-ANOVA un-ligated vs. ligated. §p < 0.05 ANOVA main effect young vs. old. Values are mean ± SEM.

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  Figure 5. Young and old vascular smooth muscle cell proliferation in response to palmitate or conditioned media from palmitate treated endothelial cells. In vascular smooth muscle cells (VSMCs) collected from young (A,B) and old (C,D) mice, proliferation was measured by Electric Cell-substrate Impedance Sensing (ECIS), where an increase in impedance suggests proliferation of cells. 48 hours after seeding (time = 0 hr on figures), VSMCs were treated directly with 100 µM palmitate or vehicle control for 72 hr (A,C). An additional set of VSMCs were treated with conditioned media from cultured endothelial cells (HUVEC) exposed to palmitate at 100, 200, or 400 µM or with vehicle control (B,D) for 72 hr. (A–D) Are the impedance over time starting at seeding (−48 hr) and continuing through the treatment period (0–72 hr). (E,F) Are summary data for the change in impedance from the start of treatment (% change from 0 to 72 hours). n = 3/group. *p < 0.05 ANOVA main effect of treatment. †p < 0.05 ANOVA main effect of age, ‡p < 0.05 ANOVA interaction of treatment x age. Values are mean ± SEM.