Our study reveals that total, but not partial, Nrf2 deficiency protects against atherosclerosis in a sex-dependent manner. We generated Nrf2 KO mice in the apoE-null background to test our hypothesis that Nrf2 deficiency would result in greater atherosclerosis because of increased tissue oxidative stress and vascular inflammation. These mice were fed a chow diet to more closely mimic human lipoprotein levels. However, contrary to our hypothesis, chow-fed male KO mice exhibited decreased atherosclerotic lesion formation with decreased macrophage content. Our results are in agreement with those of Sussan et al,27
who used other Nrf2−/−
mice fed a high-fat diet. Several differences in the experimental designs may be responsible for several contrasting effects, shown in detail in supplemental Table II
. In contrast to this previous work, Nrf2 effects on atherosclerotic lesions, inflammation, and plasma lipoproteins were sex dependent and most prominent in males in our study (supplemental Table III
To gain a better understanding of the mechanism(s) by which Nrf2 deficiency protects against atherosclerosis in males, we examined several factors known to contribute to the disease and compared their effect in males versus females and in heterozygotes versus homozygotes. Of all factors evaluated, the levels of plasma lipoproteins most closely resembled the sex- and genotype-dependent pattern exhibited by atherosclerotic lesion formation (). Our study strongly suggests an important role for decreased cholesterol levels in the decreased atherosclerosis noted in male KO mice. KO mice exhibited markedly lower plasma total and non-HDL cholesterol compared with HET and WT littermates, effects that were most pronounced in males and that resulted in positive correlations between aortic atherosclerotic scores and both plasma total cholesterol () and non-HDL/HDL cholesterol ratios (supplemental Figure VI
) in males but not in females. Moreover, KO mice exhibited lower hepatic cholesterol content than WT controls, which was also more pronounced and significant in males (P=
0.007) than in females (P=
In both sexes, Nrf2 deficiency led to reduced expression of Me1 and lipin-1, indicating that Nrf2 may have a role in the regulation of lipogenic genes (). Me1 participates in the regeneration of pyruvate from malate back to the mitochondria and assists with the release of acetyl–coenzyme A and NADPH from the mitochondria into the cytosol. Decreased Me1 could lead to lower NADPH content, resulting in decreased lipogenesis and gluconeogenesis, with lower levels of total cholesterol and glucose, as shown by Me1−/−
Lipin-1 is a phosphatase enzyme that converts phosphatidate to diacylglycerol during triglyceride and biosynthesis of phospholipids.31
However, the fact that both enzymes were reduced in KO livers from both sexes makes it less likely that they are responsible for the sex-dependent effects on atherosclerosis. Interestingly, hepatic DGAT1, responsible for the final step of triglyceride synthesis, was reduced in male mice only. Although changes in DGAT1 expression cannot explain the increased hepatic triglyceride content observed in KO mice (supplemental Figure IX
), the apparent dissociation between liver and plasma triglyceride levels could respond to sex-specific effects on the release of triglycerides to the circulating lipoproteins. In addition, DGAT1 expression levels could play a role in the sex-dependent effects noted on hepatic MCP-1 levels. Indeed, DGAT1 protects against the lipotoxic effects of fatty acids in skeletal muscle32
leading to reduced inflammation. Thus, lower DGAT1 levels in male, but not in female, KO livers could have resulted in decreased protection against fatty acid toxicity and increased expression of inflammatory markers, such as MCP-1, which occurred only in male KOs.
We also explored whether changes to macrophages might explain the decrease in atherosclerosis. Male KO mice, compared with WT controls, exhibited evidence of greater ROS generation and expression of proinflammatory cytokines in macrophages and the liver ( and supplemental Figure III
), consistent with previous studies.19,34
These findings were less pronounced or even absent in females (supplemental Figure IV and Table III
). However, these results did not explain the surprising decrease in atherosclerosis in male mice because increases in cytokines and ROS generation have been associated with increased atherosclerosis in several mouse models.
The lower macrophage area in KO atherosclerotic plaques raised the possibilities of decreased monocyte migration and/or retention in the aorta, decreased inflammatory/immune status, and accelerated removal of lesional macrophages by apoptosis or macrophage emigration. However, our data suggest that none of these are likely to be responsible for the reduced atherosclerosis in male KO mice. Thus, male KO macrophages exhibited increased MCP-1 expression () and Nrf2−/−
mice have developed strong inflammatory and immune responses against both endogenous18
stimuli. Although KO peritoneal macrophages were more susceptible to ROS-inducing conditions, such as a treatment with cadmium chloride (supplemental Figure X
), we did not observe significant differences in the degree of apoptosis in atherosclerotic lesions from KO versus WT mice by the TUNEL assay (data not shown), making it less likely that differences in the degree of apoptosis accounted for the plaque composition changes noted in vivo. Alternatively, KO mice could have exhibited a greater degree of macrophage efflux from their atherosclerotic plaques than WT littermates, as reported in a robust model of plaque regression35
; however, this may be less likely to occur in early fatty streaks under conditions of increased ROS formation, as observed in KO mice. Last, the decreased macrophage-stained area in KO lesions could have been the result of decreased size of lesional macrophages because of decreased lipid loading. Indeed, Nrf2 deficiency resulted in decreased cholesterol influx in macrophages, which correlated with lower CD36 expression. This was consistent with similar reports.27,36
However, because Nrf2 effects on lipid loading were sex independent and HET macrophages exhibited an intermediate degree of lipid loading between KO and WT cells (), it is clear that Nrf2 effects on CD36 expression and cholesterol influx can only partly explain its overall effects on atherogenesis. Instead, a significant upregulation of scavenger receptor A in KO macrophages was observed. This scavenger receptor A protein could be less active, perhaps by posttranslational modifications, such as phosphorylation,37
or its upregulation may be offset by the downregulation of CD36 or other pathways mediating decreased cholesterol influx. Thus, changes to macrophages in KO mice did not explain the difference in atherosclerosis between males and females.
Although both the present study and the study by Sussan et al27
show a decrease in atherosclerosis in Nrf2-null mice, effects on cholesterol levels were not seen in their study and there was no evidence of a preferential effect on male mice. One possible reason for this difference is the feeding of a high-fat diet that strongly elevates plasma cholesterol levels, which might have obscured the role of Nrf2 in the regulation of lipid metabolism. The study by Sussan et al emphasized an effect of Nrf2 KO on macrophage cholesterol influx. We also observed this effect, but it was not sex specific, as previously discussed. An important caveat in our studies of peritoneal macrophages and liver is that, although they are informative, they did not provide a direct assessment of changes occurring in the vascular wall. Thus, it remains possible that other elements beyond those analyzed herein may contribute to the observed outcomes.
In conclusion, Nrf2 expression results in the promotion of atherosclerosis in a sex-dependent manner, likely by a combination of systemic metabolic and local vascular effects. Our study revealed important new effects of Nrf2 that may involve the regulation of lipid metabolism, particularly plasma cholesterol levels. These effects may need to be considered when using antioxidant therapies that elevate Nrf2 expression.