The vast majority of atherosclerotic lesions, even those that are relatively large, are clinically silent (Libby, 2000
). Rather, acute atherothrombotic vascular disease, the leading cause of death in the industrialized world, is caused by a very small minority of lesions that are characterized not by large lesion volume but by plaque necrosis, plaque disruption, and susceptibility to acute thrombosis (Libby, 2000
; Virmani et al., 2003
). This report provides evidence in vitro
and in vivo
that a pathway involving the pattern recognition receptors CD36 and TLR2/6 plays an important role in a critical process leading to plaque necrosis, namely, the triggering of apoptosis in ER-stressed macrophages (Figure S3
). Although these pattern recognition receptors have been implicated previously in very different processes related to early atherogenesis (below), their role in the late lesional event of apoptosis in ER-stressed macrophages provides a link of these receptors to the completely distinct and clinically relevant process of plaque necrosis. Moreover, these studies have revealed the surprising and fascinating finding that Lp(a), a oxPL-carrying lipoprotein specifically associated with acute atherothrombosis through an unknown mechanism, is a potent stimulator of this CD36-TLR2 apoptosis pathway in ER-stressed macrophages.
While this is the first study to examine the role of CD36 and TLR2 in processes related specifically to plaque necrosis, other studies have examined their roles in early atherogenesis. For example, several studies have shown that SRA and/or CD36 deficiency protects mice from macrophage foam cell formation in mouse models of atherosclerosis, although the magnitude of this effect can be quite modest and shows dependence on the level of oxLDL-associated CD36 ligands induced by certain diets (Silverstein, 2009
; Zhao et al., 2005
; Manning-Tobin et al., 2009
; Kennedy et al., 2009
). CD36 has also been implicated in macrophage trapping in lesions (Park et al., 2009
). Similarly, a number of previous studies have examined the role of TLR2 on atherogenic processes relevant mostly to early lesion development (Mullick et al., 2005
; Mullick et al., 2008
; Liu et al., 2008
). These studies have revealed that endogenous TLR2 ligands may be acting primarily on endothelial cells, while exogenous ligands exert their effect on macrophages (Mullick et al., 2005
). Of interest, one study linked TLR2 to activation of NF-κB and the induction of inflammatory cytokines and matrix metalloproteases (MMPs) in mixed vascular cells cultured from human carotid atherectomy specimens (Monaco et al., 2009
). If future in vivo
studies provide evidence for TLR2-induced inflammation and MMP induction in advanced lesions in vivo
, these actions of TLR2 would complement the pro-apoptotic role for TLR2 revealed here in terms of advanced plaque progression.
The identity of the molecular components on oxPLs, including oxPLs associated with Lp(a)/apo(a), that activate the CD36-TLR2/6 pathway remains to be fully elucidated. Our data thus far suggest that with free oxPLs, the sn2
oxidized fatty acid is a required element, but, in view of our data showing that free saturated fatty acids (SFAs) and dipalmitoyl PC can trigger the pathway, it may work in concert with the sn1
SFA of oxPLs. However, the situation with the oxPLs associated with apo(a) is likely to be different if the sn2
oxidized fatty acid is covalently bound to the protein, as suggested by previous work (Edelstein et al., 2003
; Bergmark et al., 2008
). In this case, the sn1
SFA may play a critical role, perhaps in concert with the phosphocholine headgroup. Another area for future investigation is to determine why oxPL internalization is necessary to trigger apoptosis in ER-stressed macrophages (Figure S5
) and how this process might work in concert with cell-surface signaling through CD36 and/or TLR2/6 heterodimer.
The role of SFAs in the apoptosis pathway described here is an important finding both mechanistically and because of its potential relevance to obesity and insulin resistance. SFAs have been reported to directly stimulate TLR signaling (Shi et al., 2006
; Senn, 2006
; Nguyen et al., 2007
), but these findings have recently been challenged by a study showing LPS contamination in the BSA used to complex the SFAs (Erridge and Samani, 2009
). Contamination of BSA with TLR activators cannot account for the ability of SFAs to trigger apoptosis in ER-stressed macrophages because LPS in our model does not serve as a second TLR hit for apoptosis in ER-stressed macrophages (Seimon et al., 2006
), and the pro-apoptotic effect of fatty acids in the current study was TLR4-independent. Moreover, the SFA-free BSA control did not trigger macrophage apoptosis when combined with thapsigargin, and SFAs were much more potent in inducing apoptosis than unsaturated FAs, which were also complexed with BSA. In addition to activating TLRs, SFAs can directly trigger pro-apoptotic ER stress (Wei et al., 2006
), which likely accounts for the residual level of TLR2-independent macrophage death we observed with SFAs in the absence of thapsigargin (). In this regard, a recent study showed that SFAs at a dose of 500 μM alone can cause ER stress-induced macrophage death through a pathway mediated by adipocyte-binding protein-4 (aP2), a cytosolic lipid chaperone that is also expressed in macrophages (Erbay et al., 2009
). However, our in vivo
studies with ob/ob
mice suggest that the TLR-dependent pathway of macrophage death revealed herein is relevant to pathological settings in which ER stress is present.
As mentioned above, our study may provide insight into how Lp(a) contributes to atherothrombotic vascular disease. Lp(a) is an independent risk factor for coronary artery disease, but the mechanism underlying this observation is not known (Tregouet et al., 2009
; Brazier et al., 1999
; Holmer et al., 2003
; Bergmark et al., 2008
; Erqou et al., 2009
). Recent insight was provided by the finding that Lp(a) functions as a carrier of PC-containing oxPLs (Bergmark et al., 2008
), and we show here that oxPL-containing Lp(a) and apo(a) can trigger apoptosis in ER-stressed macrophages in a CD36-, TLR2/6-, ROS-, and sn2
fatty acid-dependent manner. These findings may explain a report in 1994 showing that Lp(a) can induce oxidative stress in monocytes (Riis Hansen et al., 1994
), although no mechanism was reported. Relevant to a role for Lp(a) in ER stress-induced macrophage apoptosis, transgenic hyperlipidemic rabbits with modest plasma levels of Lp(a) have a marked increase in plaque necrosis without a change in plasma lipids or en face
lesion area compared with non-transgenic WHHL rabbits (Sun et al., 2002
). This finding is consistent with the association between high plasma levels of Lp(a) and acute coronary syndromes. Thus, our findings raise the possibility of a specific cellular mechanism linking elevated plasma Lp(a) to advanced plaque progression and consequent atherothrombotic vascular disease.
The ultimate goal of studies on advanced plaque progression in general, and advanced lesional macrophage apoptosis in particular, is to suggest therapeutic strategies that specifically block the progression of the 2–3% of total human atherosclerotic lesions that actually cause acute atherothrombotic vascular disease. If one were to focus on interrupting the proximal components of the pathway described herein, i.e.
, CD36 or TLR2, lesional targeting would likely be required for specificity in view of the widespread role of these receptors in normal physiology and host defense. A further therapeutic clue from the data herein may come from the critical role of prolonged oxidative stress. On the one hand, we show here that ER stress is needed for prolongation of this response per se
, and recent work using chemical chaperones to suppress ER stress has suggested potential promise of this approach in the setting of atherosclerosis (Erbay et al., 2009
). The initial oxidant burst itself, which is triggered by a TLR2-NADPH oxidase pathway, is also a possible target through NADPH oxidase inhibitors (Yu et al., 2008
) or anti-oxidants that partially suppress the response but do not compromise its role in host defense. The fact that vitamin E is not useful in preventing human cardiovascular disease may be due to the fact that it does not adequately target the specific types and mechanisms of oxidative stress that are key to plaque progression (Williams and Fisher, 2005
). Indeed, the protective actions of NAC in our cell culture model of ER stress-induced macrophage apoptosis were not found with doses of vitamin E that have anti-oxidant actions in other settings (unpublished data). Of interest in this regard, NAC has been shown recently to suppress atherosclerosis in fat-fed Apoe
−/− mice (Shimada et al., 2009
). These types of findings and therapeutic strategizing provide strong rationale for understanding in depth the cellular and molecular mechanisms that specifically promote advanced plaque progression.