The major finding of this study is that Foxo4-deficiency in vivo enhances atherosclerotic lesion burden in a mouse model of atherosclerosis (). Our studies also suggest that the cellular mechanism by which FoxO4 inhibits atherosclerosis is likely through its function in monocyte from bone marrows, as we have shown that Foxo4-inactivation in bone marrow derived cells is sufficient to promote the development of atherosclerosis and to upregulate the macrophage content in the lesion (). How does FoxO4 inhibit atherosclerosis through its function in monocytes/macrophages?
Macrophages in the atherosclerotic lesion are mainly derived from circulating blood monocytes that have undergone differentiation upon activation by modified lipids and/or lipoproteins. The upregulation of macrophages in lesions of HFD-fed Foxo4
-null mice could result from the increased recruitment of monocytes. FoxO proteins are known to activate the transcription of several ROS scavenge enzymes, including manganese superoxide dismutase, catalase, and peroxiredoxin 3 (Chiribau et al., 2008
, Keizer al., 2010
). ROS are essential for normal metabolism, but they are potentially destructive if not tightly controlled (Harrison et al., 2003
). Excessive ROS have been shown to be involved in several atherosclerotic processes, including LDL oxidation, alteration of endothelial cell function, macrophage trapping in the lesion, and the stimulation of VSMC proliferation and migration (Souza et al., 2003
; Park et al., 2009a
, Tabas, 2010
). Inactivation of Foxo4
could result in excess of ROS that promotes EC dysfunction, which in turn enhances monocyte recruitment. To test this hypothesis, we measured the ROS level of WT and Foxo4
-null macrophages treated with and without LPS, and tested the adhesion of activated/non-activated WT and Foxo4
-null macrophages to HUVECs. Although Foxo4
-null macrophages produced higher level of ROS upon stimulation by LPS when compared to wild type macrophages (), activated Foxo4
-null macrophage adhesion to HUVECs is similar to that of WT macrophages (data not shown), suggesting that increased production of ROS from Foxo4
-null macrophage alone may not be sufficient to promote its recruitment by ECs.
FoxO4 could also inhibit atherosclerosis through its action on immune cell homeostasis. Previously, we found that FoxO4 plays an important role in mucosal innate immunity through regulating expression of NF-κB-activated inflammatory cytokines, including CCL5, CXCL9, TNFα, IFNγ, IL-1β, and IL-6 (Zhou et al., 2009
). As NF-κB plays a central role in the process of vascular inflammation (Brasier, 2010
; Gareus et al., 2008
; Van der Heiden et al., 2010
), it is possible that FoxO4-regulated immune response may play a role in atherosclerosis. Indeed, atherosclerotic lesions in HFD-fed Foxo4
-deficient mice, either Foxo4/apoE
DKO mice or chimeric mice with Foxo4
-null bone marrows, have increased amount of immune cells, indicating an escalation of local inflammation. Furthermore, circulating IL-6 levels in HFD-fed Foxo4/apoE
DKO mice were elevated compared to those of apoE
SKO mice, suggesting an elevation of systemic inflammation. Chemokines and inflammatory cytokines secreted by Foxo4
-null immune cells could promote dedifferentiation and proliferation of residence smooth muscle cells and dysfunction of endothelial cells, which could in turn enhance further recruitment of immune cells and perpetuate the inflammation.
The atherogenic diet we used in this study contains cholic acid. The presence of cholic acid has been shown to aid cholesterol and fat absorption, and to suppress conversion of cholesterol to bile acids (Ando et al., 2005
), which reduces removal of cholesterol and promotes early formation of atherosclerosis in susceptible mouse strains such as C57BL/6 (Nishina et al., 1990
). Cholic acid has also been shown to influence transcription factors in transcriptional regulation of genes involved in lipid metabolism and inflammation (Ander et al., 2005). Whether cholic acid is involved in FoxO4-regulated gene transcription remains to be determined.
In this study, we found that chimeric C57B/6 mice reconstituted with Foxo4-null bone marrows have enhanced atherosclerosis in the aortic root than mice received with wild-type bone marrows. This finding is significant as it shows that inaction of Foxo4 alone in bone marrow derived cells is sufficient to promote regional atherosclerosis even in the absence of a defect in lipid metabolism such as inactivation of apoE, demonstrating the critical importance of the immune component of atherogenesis. Nonetheless, a reciprocal experiment with the Foxo4-null mouse as the recipient and WT bone marrow as the donor should further clarify the role of Foxo4-null bone marrow in atherogenesis.
It is also interesting to note that the lesions were found only in the aortic root in C57B/6 mice after feeding with HFD. No lesions were observed in the descending aorta. This is expected, as it was known that flow characteristics is a risk factor for development of atherosclerosis and the aortic root area is prone to atherosclerosis due to its disturbed flow (Traub & Berk, 1998
; Haidari et al., 2010
). Consistent with the observation that only localized atherosclerotic plaques are upregulated in the chimeric mice with Foxo4
-null bone marrows compared with chimeric mice with WT bone marrows, markers of systemic inflammation such as serum IL-6 were not significantly upregulated in these mice either (data not shown), suggesting that inactivation of Foxo4 in bone marrows may not be sufficient to cause systemic inflammation. These results also suggest the importance of non-immune cell components such as ECs and SMCs in the vascular wall in the initiation of atherogenesis. At present, it is not clear which cell type in the vascular wall is the main target for FoxO4-induced vascular inflammation. Further studies are needed to identify the vascular cell types that mediate the effect of FoxO4 on atherosclerosis.
Previously, we showed that FoxO4 promotes dedifferentiation and proliferation of arterial SMCs upon mechanical injury, as inactivation of Foxo4
led to a decreased neointimal formation upon carotid artery ligation (Li et al., 2007
). The amount of SMCs in HFD-fed Foxo4/apoE
DKO was not significantly upregulated compared to that of apoE
SKO mice (data not shown), suggesting that the effect of FoxO4 on SMC proliferation may be context-dependent. This is not surprising, since the signals that act upon vascular SMCs in atherosclerosis are ROS and cytokines whereas those in the carotid artery ligation model are characteristics of flow-induced mechanical forces.
In the current study, we also observed increased amount of T-cells in atherosclerotic lesions of Foxo4/apoE
DKO mice compared to apoE
SKO mice. This is significant as T-cell mediated adaptive immunity is known to drive the progression of atherosclerosis (Hansson & Jonasson, 2009
; Anderson et al., 2010
). It will be worthy to determine the mechanism(s) by which Foxo4
-deficiency leads to the upregulation of T-cells in the atherosclerotic lesion of Foxo4/apoE
DKO mice. IL-6 is a T-cell survival factor and promotes expansion of effect/memory T-cells (Dienz & Rincon, 2009
). The increased amount of CD4+ T cells in the lesions of HFD-fed DKO mice could be the consequence of increased production of IL-6 in the lesion. Alternatively, FoxO4 could have T-cell autonomous functions similar to that of FoxO1 (Fabre et al., 2008
; Dengler et al., 2008
). A mouse model with Foxo4
-inactivated in the T-cell compartment could facilitate our understanding of the role of FoxO4 in T-cell mediated atherosclerosis in the future.
In conclusion, our studies indicate that FoxO4 is an anti-atherogenic factor and inhibits atherosclerosis through its function in bone marrow derived monocyte/macrophages. FoxO proteins are major downstream effectors of insulin-activated PI3K/Akt signaling pathway. Inactivation of the PI3K/Akt signaling pathway activates FoxO proteins and promotes longevity in worms (Kenyon, 2010
) and flies (Puig & Tijin 2006
; Lee et al., 2009
). Our data suggest that activation of FoxO proteins through the insulin/Akt pathway could also be beneficial in inhibition of atherosclerosis and maintaining a long and healthy life-span in humans.