Objective-To determine if cannabinoid receptor 2 (CB2) plays a role in atherosclerosis, we investigated the effects of systemic CB2 gene deletion on hyperlipidemia-induced atherogenesis in low density lipoprotein receptor-deficient (Ldlr −/− ) mice.Methods and results-Ldlr −/− and CB2/Ldlr double knockout (CB2 −/− Ldlr −/− ) mice were fed an atherogenic diet for 8 and 12 weeks. Morphometric analysis revealed no significant difference between the atherosclerotic lesion area in the proximal aortas of Ldlr −/− and CB2 −/− Ldlr −/− mice after 8 or 12 weeks on the atherogenic diet. The macrophage and smooth muscle cell (SMC) content, as revealed by immunohistochemical staining, did not differ significantly between Ldlr −/− and CB2 −/− Ldlr −/− lesions after 8 weeks. However, after 12 weeks, CB2 −/− Ldlr −/− lesions displayed greater macrophage content (86.6 ± 4.1 versus 75.2 ± 7.5%, P < 0.05) and SMC content (11.1 ± 5.1 versus 4.2 ± 2.4%, P < 0.05) compared to controls. Lesional apoptosis, as determined by in situ TUNEL analysis, was reduced ∼50% in CB2 −/− Ldlr −/− lesions after 12 weeks. CB2 −/− Ldlr −/− lesions displayed significantly reduced collagen content and increased elastin fiber fragmentation after 12 weeks, which was associated with an ∼57% increase in matrix metalloproteinase 9 (MMP) levels. In vitro, CB2 −/− macrophages secreted ∼1.8-fold more MMP9 activity than CB2 +/+ macrophages. Conclusions-CB2receptor deficiency affects atherogenesis in Ldlr-null mice by increasing lesional macrophage and SMC content, reducing lesional apoptosis and altering extracellular matrix components, in part, by upregulating MMP9. These results suggest that pharmacological manipulation of CB2 receptors might exert multiple and complex effects on atherogenesis and plaque stability.

Macrophage apoptosis is an important process in the pathophysiology of atherosclerosis. Oxidized lowdensity lipoproteins (OxLDL) are a major component of lesions and potently induce macrophage apoptosis. Cannabinoid receptor 2 (CB2), the predominant macrophage cannabinoid receptor, modulates several macrophage processes associated with ongoing atherosclerosis; however, the role of CB2 in macrophage apoptosis is unknown. To determine if CB2 influences a macrophage apoptotic pathway relevant to atherosclerosis, we examined the effect of CB2 deficiency on OxLDL-induced macrophage apoptosis. In situ terminal transferase-mediated dUTP nick end labeling (TUNEL) analysis of resident peritoneal macrophages detected significantly fewer apoptotic CB2 2/2 macrophages than CB21/1 macrophages after incubation with OxLDL (27.9 6 4.7% vs. 61.9 6 8.5%, P , 0.001) or 7-ketocholesterol (7KC) (18.9 6 10.5% vs. 54.1 6 6.9%, P , 0.001), an oxysterol component of OxLDL. Caspase-3 activity; proteolytic conversion of procaspase-3; and cleavage of a caspase-3 substrate, PARP, were also diminished in 7KC-treated CB2 2/2 macrophages. Furthermore, the deactivation of the prosurvival kinase, Akt, in response to 7KC was impaired in CB2 2/2 macrophages. These results suggest that CB2 expression increases the susceptibility of macrophages to OxLDLinduced apoptosis, in part, by modulating the effect of oxysterols on the Akt survival pathway and that CB2 may influence atherosclerosis by modulating lesional macrophage apoptosis.

Oxysterol-induced macrophage apoptosis may have a role in atherosclerosis. Macrophages lacking the type 2 cannabinoid receptor (CB2) are partially resistant to apoptosis induced by 7-ketocholesterol (7KC). AM-251 and SR144528 are selective antagonists of CB1 and CB2 receptors, respectively. We observed that both compounds reduce 7KC-induced apoptosis in Raw 264.7 macrophages. As oxysterol-induced macrophage apoptosis requires acyl-coenzymeA:cholesterol acyltransferase (ACAT) activity, we tested their affects on ACAT activity. AM-251 and SR144528 both reduced cholesteryl ester synthesis in unstimulated and acetylated LDL-stimulated Raw 264.7 macrophages, CB2 +/+ and CB2 −/− peritoneal macrophages, as well as in vitro, in mouse liver microsomes. Consistent with inhibition of ACAT, the development of foam cell characteristics in macrophages by treatment with acetylated LDL was reduced by both compounds. This work is the first evidence that AM-251 and SR144528 are inhibitors of ACAT and as a result, might have antiatherosclerotic activities independent of their affect on cannabinoid signaling. Keywords 7-ketocholesterol; ACAT; apoptosisDuring atherogenesis, macrophages in the vascular intima take up modified low density lipoproteins (LDL), such as oxidized LDL (OxLDL), and store much of the LDL-derived cholesterol as cholesteryl esters in cytosolic lipid droplets [1]. Accumulation of cytosolic lipid droplets within macrophages transforms them into the foam cells which define early atherosclerotic lesions known as fatty streaks [2]. Intracellular cholesteryl ester synthesis is carried out by acyl-coenzyme A:cholesterol acyltransferase (ACAT) using cholesterol and fatty-acyl CoA as substrates [3]. Inhibition of ACAT prevents foam cell formation and, in some animal studies, slows the progression of atherosclerosis [4].Lesional macrophage apoptosis plays several roles during the pathophysiology of atherosclerosis. During early lesion formation, macrophage apoptosis is anti-atherosclerotic [5,6] while at later stages it likely contributes to plaque destabilization [7]. Lesional macrophages are subject to several apoptotic inducers, including oxLDL, which potently induces apoptosis in cultured macrophages largely as a result of its cytotoxic oxysterol *Corresponding author: Phone: 423-439-2131, Fax: 423-439-2130, Email: Thewke@etsu.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [8,9]. Oxysterols are also substrates for ACAT and reduction of ACAT activity in macrophages produces resistance to 7KC-induced apoptosis, indicating that ACAT is a required step in the signal...

Akt plays a role in protecting macrophages from apoptosis induced by some oxysterols Previously we observed enhanced degradation of Akt in P388D1 moncocyte/macrophages following treatment with 25-hydroxycholesterol (25-OH) or 7-ketocholesterol (7-KC). In the present report we examine the role of the ubiquitin proteasomal pathway in this process. We show that treatment with 25-OH or 7-KC results in the accumulation of poly-ubiquitinated Akt, an effect that is enhanced by cotreatment with the proteasome inhibitor MG-132. Modification of Akt by the addition of a Gly-Ala repeat (GAr), a domain known to block ubiquitin-dependent targeting of proteins to the proteasome, resulted in a chimeric protein that is resistant to turn-over induced by 25-OH or 7-KC and provides protection from apoptosis induced by these oxysterols. These results uncover a new aspect of oxysterol regulation of Akt in macrophages; oxysterol-stimulated poly-ubiquitination of Akt and degradation by the proteasomal pathway.