Gain and loss of function for glutathione synthesis: impact on advanced atherosclerosis in apolipoprotein E-deficient mice

Abstract

Objective: Glutamate-cysteine ligase (GCL) is the rate-limiting step in glutathione synthesis. The enzyme is a heterodimer composed of a catalytic subunit, GCLC, and a modifier subunit, GCLM. We generated apolipoprotein E (apoE)-/- mice deficient in GCLM (apoE-/-/Gclm-/-) and transgenic mice that overexpress GCLC specifically in macrophages (apoE-/-/Gclc-Tg) to test the hypothesis that significantly altering the availability of glutathione has a measurable impact on both the initiation and progression of atherosclerosis.

Methods and results: Atherosclerotic plaque size and composition were measured in the innominate artery in chow-fed male and female mice at 20, 30, 40, and 50 weeks of age and in the aortic sinus at 40 and 50 weeks of age. The apoE-/-/Gclm-/- mice more rapidly developed complex lesions, whereas the apoE-/-/Gclc-Tg mice had reduced lesion development compared with the littermate apoE-/- control mice. Transplantation of bone marrow from the apoE-/-/Gclm-/- and apoE-/-/Gclc-Tg mice into apoE-/- mice with established lesions also stimulated or inhibited further lesion development at 30 weeks posttransplant.

Conclusion: Gain and loss of function in the capacity to synthesize glutathione especially in macrophages has reciprocal effects on the initiation and progression of atherosclerosis at multiple sites in apoE-/- mice.

Figure 1. Average Lesion Area in the Innominate Artery and Aortic Sinus

ApoE−/−/Gclm−/− Mice (A) Male mice at 20 weeks (apoE−/−/Gclm−/− n=5, apoE−/−/Gclm+/+ n=17), 30 weeks (apoE−/−/Gclm−/− n=8, apoE−/−/Gclm+/+ n=13), 40 weeks (apoE−/−/Gclm−/− n=7, apoE−/−/Gclm+/+ n=10) and 50 weeks (apoE−/−/Gclm−/− n=9, apoE−/−/Gclm+/+ n=4); (C) female mice at 20 weeks (apoE−/−/Gclm−/− n=5, apoE−/−/Gclm+/+ n=24), 30 weeks (apoE−/−/Gclm−/− n=8, apoE−/−/Gclm+/+ n=14), 40 weeks (apoE−/−/Gclm−/− n=6, apoE−/−/Gclm+/+ n=10) and 50 weeks (apoE−/−/Gclm−/− n=5, apoE−/−/Gclm+/+ n=20). (E) Aortic sinus of female mice at 40 weeks of age (apoE−/−/Gclm−/− n=5, apoE−/−/Gclm+/+ n= 5) ApoE−/−/Gclc-Tg Mice. (B) Male mice at 20 weeks (apoE−/−/Gclc-Tg n=11, apoE−/−/Gclc-WT n=12), 30 weeks (apoE−/−/Gclc-Tg n=7, apoE−/−/Gclc-WT n=6), 40 weeks (apoE−/−/Gclc-Tg n=6, apoE−/−/Gclc-WT n=11) and 50 weeks (apoE−/−/Gclc-Tg n=7 apoE−/−/Gclc-WT n=13); (D) Female mice at 20 weeks (apoE−/−/Gclc-Tg n=14, apoE−/−/Gclc-WT n=15), 30 weeks (apoE−/−/Gclc-Tg n=15, apoE−/−/Gclc-WT n=12), 40 weeks (apoE−/−/Gclc-Tg n=7, apoE−/−/Gclc-WT n=7) and 50 weeks (apoE−/−/Gclc-Tg n=8, apoE−/−/Gclc-WT n=18); (F) Aortic sinus of male mice at 50 weeks of age (apoE−/−/Gclc-Tg n=5, apoE−/−/Gclc-WT n=5). Data are presented as means ± SE, *p<0.05 vs controls.

Figure 2. Average Lesion Area in the Innominate Artery Following Bone marrow Transplant

ApoE−/−/Gclm−/− Mice. A) Lesion area in female apoE−/− mice following bone marrow transplantation at 20 weeks (apoE−/−/Gclm−/− donor, apo E−/− recipients n=15, apoE−/−/Gclm+/+ donor, apo E−/− recipients n =14), and 30 weeks (apoE−/−/Gclm−/− donor, apo E−/− recipients n=13, apoE−/−/Gclm+/+ donor, apo E−/− recipients n=8). ApoE−/−/Gclc-Tg Mice. B) Lesion area in female apoE−/− mice following bone marrow transplantation at 20 weeks post-transplant (apoE−/−/Gclc-Tg donor, apo E−/− recipients n=15, apoE−/−/Gclc-WT donor, apo E−/− recipients n=14), and 30 weeks post-transplant (apoE−/−/Gclc-Tg donor, apo E−/− recipients n=13, apoE−/−/Gclc-WT, apo E−/− recipients donor n=8). Data are presented as means ± SE, *p<0.05 vs controls. All recipient apo E−/− mice were from the same breeding colony as the apo E−/− mice used to generate the apo E−/−/Gclc-Tg mice.

Figure 3. Macrophage Content of the Lesions in the Innominate Artery

Area of Mac 2 staining in lesions in the innominate artery as a percentage of the total area of the lesion for males (A, B), and females (C, D). Sample sizes were identical to those listed in figure 1. Data shown are the means ± SE. * p<0.05 vs littermate control.

Figure 4. Total Cell Content of the Lesions in the Innominate Arteries and Caspase-9 Activity in Peritoneal Macrophages

Total # nuclei/1000 μm² cross sectional area in lesions in the innominate artery of both the male (A,C) and female (B,D) mice. Sample sizes were identical to those listed in figure 1. Caspase 9 activity (E,F) was evaluated in thioglycollate-elicited peritoneal macrophages after 6h treatment with acrolein or staurosporin (n= 3 mice/group). *p<0.05 vs littermate controls. Data are presented as means ± SE.