The data in this study reveal 2 important points about the function of SR-A and CD36 in advanced aortic root atherosclerotic lesions of Apoe−/−
mice. First, the receptors are not absolutely necessary for foam cell formation in a markedly hyperlipidemic animal, nor do they appear to contribute substantially to overall lesion size, despite the fact that they can account for the vast majority of oxidized LDL uptake in cultured macrophages. Second, the receptors contribute to inflammatory gene expression and appear to play an important role in the generation of advanced lesional macrophage apoptosis and plaque necrosis. These data are consistent with recent mechanistic studies from our laboratories linking scavenger receptors to downstream signaling pathways and their role in precipitating macrophage apoptosis.21,23,27–29
Since the first report of reduced atherosclerosis in Apoe−/−
mice lacking SR-A by Suzuki and colleagues in 1997, the paradigm has been that scavenger receptors promote atherosclerosis primarily through their role in generating macrophage foam cells.30
However, additional studies of the role of SR-A in atherosclerosis have led to varied results, with some studies showing it to be proatherogenic and others suggesting an antiatherogenic effect.17,31–33
Studies of the impact of CD36 on atherosclerosis in Apoe−/−
mice have been some-what more consistent, with deficiency of CD36 conferring protection against atherosclerotic lesion development in the descending aorta ranging from 30% to 80%.17,33,34
A more variable effect of CD36 on aortic root lesions has been reported, however these effects were modest compared with the benefit conferred on more distal aortic lesions.17,34
Thus, CD36 may evince region-specific influences on atherosclerosis. A key finding of the combined SR-A/CD36 knock-out study reported here is that the failure of prior studies of mice lacking either SR-A or CD36 to demonstrate elimination of aortic root foam cell formation cannot now be explained by compensation by the other receptor. While our study was being completed, Kuchibhotla et al reported that Apoe−/−
mice lacking both SR-A and CD36, fed a western diet for 12-weeks, also retained foam cell formation in their aortic root lesions.33
Taken together, these data now clearly establish that foam cell formation can proceed in the absence of these scavenger receptor pathways.
How might foam cells form in vivo in the absence of SR-A and CD36? Other receptors such as scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX/ CXCL16) or lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) may internalize oxidized lipoproteins, and an increase in their expression could compensate for the loss of SR-A and CD36. We performed quantitative RT-PCR analysis of the expression of both SR-PSOX and LOX-1 in RNA taken from the aortas of female wild-type and TKO mice, but were unable to detect significantly increased expression for either receptor, using 4 to 5 animals per assay (data not shown). Foam cell formation can also be driven in cultured macrophage models using LDL that has not been oxidized (reviewed in35
). Early studies by Khoo et al and Tabas and colleagues showed that aggregated LDL, known to be present in atherosclerotic lesions, is a potent scavenger receptor–independent inducer of foam cells in cultured macrophages.36,37
Other examples of scavenger receptor–independent foam cell–forming particles include sPLA2-modified LDL, chylomicron remnants, and platelet-derived vesicles.16,38
Furthermore, Kruth and colleagues have provided strong evidence that macrophages can internalize native lipoproteins via the process of macropinocytosis, a nonreceptor-mediated process that would likely take on increasing importance as the local concentration of lipoproteins increased.18
One important caveat of our work is that this study was conducted in Apoe−/−
mice fed a Western diet. As these mice are markedly hyperlipidemic, the role of high affinity modified lipoprotein receptor uptake mechanisms in foam cell formation might well be less important in this setting than in situations of moderate hypercholesterolemia. Thus, this work does not preclude a more substantial role for SRA and CD36 in foam cell formation in other animal models or in human atherosclerosis.
Despite only a modest impact on overall atherosclerosis lesion area, we found that scavenger receptor deficiency causes substantial changes in atherosclerosis inflammatory gene expression. Decreased aortic expression of cytokines, chemokines and adhesion molecules indicated that the loss of the scavenger receptors was associated with a reduction in the inflammatory milieu. In agreement with this, Kuchibhotla et al reported decreased serum levels of inflammatory markers in their Apoe−/− Cd36−/− Msr1−/−
mice, several of which overlap with our measurements of inflammatory gene expression in the aorta.33
In addition, we found that the combined absence of SR-A and CD36 markedly reduced both macrophage apoptosis and plaque necrosis in lesions of the aortic root. Plaque necrosis results when macrophages become apoptotic and then are not efficiently cleared by efferocytes. The observed decrease in lesional apoptosis is consistent with our previous in vitro studies showing that apoptosis in ER-stressed macrophages can be triggered by SRA and CD36 ligands (Seimon and Tabas, unpublished observations).21,23
Markers of ER stress correlate with advanced lesion stage and apoptosis in both animal and human coronary artery plaques, supporting a role for this process in triggering macrophage apoptosis and necrotic core formation.22
Moreover, gene targeting studies in mice have shown causal relationships among ER stress, advanced lesion macrophage apoptosis, and plaque necrosis.39
The finding of decreased necrotic cores in mice lacking the scavenger receptors might initially seem to be in conflict with our failure to demonstrate significant changes in lesion area or substantial differences in macrophage number in the atherosclerotic lesions. Necrotic core area, however, is a measurement that integrates events that are occurring over the entire time course of our study, and it reflects macrophage apoptosis, efferocytosis, and exit from the intima, as well as new macrophage recruitment to lesions. To fully understand the effects of the loss of scavenger receptors on lesion development and progression, it will be necessary to undertake a detailed analysis of these processes in future studies that assess these events in temporal sequence.
In summary, the data in this study demonstrate that scavenger receptors play a critical role in the evolution of atherosclerosis lesion complexity. This role appears to be mediated through their influence on apoptosis and inflammatory gene expression, rather than lipid uptake and foam cell formation. These findings also suggest that the analysis of more complex features of atherosclerosis, such as the quantification of necrotic core volumes, will increasingly be needed if we are to decipher the complex interplay between innate immune systems, apoptosis, and the evolution of the atherosclerotic plaque.