Pharmacological studies suggest that the CB2 receptor plays a role in atherosclerosis [14
]. In the current study, we directly examined the role of the CB2 receptor in atherosclerosis by investigating the effects of systemic CB2 receptor deficiency on atherosclerosis in Ldlr-null mice. We observed that CB2 receptor deficiency did not affect the size of lesions in Ldlr−/−
mice; however, it did increase lesional macrophage accumulation and SMC infiltration, in addition to reducing lesional apoptosis and altering the ECM of lesions.
The lack of an effect of CB2 receptor gene deficiency on the size of atherosclerotic lesions in Ldlr-null mice is somewhat unexpected as administration of exogenous cannabinoids decreases the size of atherosclerotic lesions in ApoE−/−
mice by mechanisms sensitive to CB2 receptor-selective antagonism [14
]. There are several possible explanations for these somewhat contradictory results. First, activation of CB2 receptors by endogenous ligands during atherogenesis may not be sufficient to significantly affect lesion size, but when exogenous cannabinoids are supplemented, CB2 receptor signaling is augmented to levels sufficient to produce an affect on lesion size. Second, a novel cannabinoid receptor, which compensates for the loss of CB2 in CB2−/−
mice, may be responsible for the effects of exogenous cannabinoid supplementation on lesion size. Pharmacological evidence for novel cannabinoid receptors, some of which display sensitivity to CB1 - and CB2-selective antagonists, lends some support to this suggestion [23
]. Lastly, these results may be due to differences between the Ldlr−/−
mouse strains, which are well-known to vary in lipoprotein characteristics and other parameters that determine the pathophysiology of atherosclerosis [25
The observation that lesional macrophage content is unaffected by CB2 receptor deficiency in Ldlr−/−
mice in lesions after 8 weeks of atherogenic diet intervention but is increased in lesions after 12 weeks indicates that CB2 receptor-mediated signaling does not play a significant role in monocyte/macrophage recruitment during initial lesion formation but instead, becomes sufficient to reduce macrophage recruitment as lesions progress. This is consistent with the prior study by Steffens et al. [14
] demonstrating that Δ9-tetrahydrocannabinol-induced lesion regression in ApoE−/−
mice was, at least partly, a consequence of reduced leukocyte recruitment. In this scenario, exogenous cannabinoid supplementation augments endogenous CB2 receptor activation resulting in a greater impairment of monocyte/macrophage recruitment.
Although no variation in the level of CB1 receptor expression in CB2-null macrophages was observed (Supplemental Fig. 1
), we can not rule out the possibility that the lack of more pronounced effects of CB2 receptor deficiency on lesion formation is not due to compensatory activation of CB1 receptors or compensatory expression of a novel cannabinoid receptor(s). Evidence of novel cannabinoid receptors in the vasculature lend some support to this suggestion [23
]. Future studies examining the effect of cannabinoid supplementation on lesional macrophage content in CB2−/−
mice should further clarify the role of CB2 receptors in cannabinoid-induced lesion regression.
Proliferation and migration of vascular SMCs are important events in atherogenesis which could be affected by CB2 receptor signaling. In vitro
, SMC proliferation and migration induced by TNF-α is attenuated by CB2 receptor agonists [26
], while SMC proliferation and migration in response to PDGF is attenuated by SR141716, a CB1 receptor antagonist [27
]. This suggests that CB1 and CB2 receptors may differentially modulate the proliferative and migratory responses of SMCs to cytokines and chemokines. The increased SMC immunoreactivity observed in CB2−/−
lesions is consistent with an inhibitory role of CB2 receptors in SMC proliferation and migration during atherosclerosis. Indirect effects brought about by the interaction between CB2-null macrophages and SMCs may also contribute to the increased SMC content of CB2−/−
lesions. The inability to ascribe the observed effects of CB2 receptor deficiency to any one particular cell type is an inherent limitation of the current experimental approach. Experiments employing adoptive transfer of CB2-deficient bone marrow derived cells in to Ldlr-null mice should provide insight into the relevant cell types involved in CB2 receptor-modulated events during atherosclerosis.
Diminished lesional apoptosis in CB2 receptor-null mice is in agreement with our prior in vitro
study demonstrating reduced susceptibility of CB2-null macrophages to oxLDL-induced apoptosis [15
], and our previous study associating reduced lesional apoptosis (due to Bax gene deficiency) with increased lesional macrophage content [3
]. It is important to note that the CB2 receptor and Bax likely play different roles in apoptosis. While Bax functions in the execution phase of apoptosis which is common to a broad range of apoptosis inducers, CB2 receptors modulate the response of macrophages to specific apoptosis inducers, the presence of which likely varies within atherosclerotic lesions as they progress. The observation that reduced apoptosis was not observed in lesions of CB2-null mice after 8 weeks on an atherogenic diet, but was evident after 12 weeks, suggest that a threshold level of a specific apoptosis inducer, and/or CB2 ligand, must accumulate before CB2-mediated modulation of apoptosis occurs [15
]. Although TUNEL-positive nuclei were largely localized to macrophage-enriched regions, it is possible that CB2 receptor deficiency also alters apoptosis of cell types in addition to macrophages in atherosclerotic lesions.
CB2 receptor deficiency could reduce collagen content by slowing collagen synthesis and/or accelerating collagen degradation. SMCs are a primary source of collagen deposition in atherosclerotic lesions [28
], however, the observed increase in SMC content in lesions of CB2-null mice suggest that collagen reduction is not due to lower numbers of intimal SMCs; although, a reduction of collagen synthesis by CB2-null SMCs can not be ruled out. The observation of increased MMP9 immunoreactivity in CB2−/−
lesions, as well as increased secretion of MMP9 activity from CB2-null macrophages, suggests that upregulation of MMP9 in CB2−/−
lesions is, at least partially, responsible for the reduction of collagen. In support of this hypothesis, MMP9 is suppressed in immune cells by cannabinoid compounds [30
] and was induced by CB2 receptor antagonism (). Increased disruption and fragmentation of medial elastic fibers in CB2−/−
lesions may also be the result of increased MMP9 activity. Increased degradation of elastin may also facilitate the migration of SMCs from the media into the intima and contribute to the increase in SMC content of CB2−/−
lesions. Collectively, these results suggest that CB2 receptor signaling contributes to the establishment and maintenance of more stable atherosclerotic lesions, in part, by suppressing MMP9. It should also be noted that while zymography reveals the total enzymatic activity, it may not reflect the net in vivo
activity of MMP9 which is subject to the levels of tissue inhibitors of matrix metalloproteinases. Future studies examining MMP activities in atherosclerotic lesions of CB2−/−
mice should clarify the role of CB2 receptors in modulating MMP activities and extracellular matrix remodeling of lesions.
Macrophage death is common in the shoulders of rupture-prone human plaques and is considered a destabilizing factor in plaque rupture [5
]. Therefore, even though degradation of ECM components may promote lesion destabilization, the absence of CB2 receptor signaling might also provide a beneficial effect due to reduced lesional apoptosis in more advanced lesions. Determining the extent to which any modulation of macrophage apoptosis associated with CB2 receptor deficiency, or treatments with CB2-selective agonists and antagonists, affects plaque stability, will require studies using ApoE−/−
mice in which plaque rupture can be dependably observed in the innominate artery [32
In conclusion, the present study provides the first direct evidence that CB2 receptor signaling is not significantly involved in the initial formation of atherosclerotic lesions, but may play an important role in modulating cellular composition and stability of lesions as they progress. These results support the idea that pharmacologic compounds with activity at CB2 receptors may be useful in modifying atherosclerosis and underscore the need for further studies aimed at more fully elucidating the role of CB2 receptor signaling in plaque composition and vulnerability.