The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.

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2-Methoxyestradiol (2-ME), an endogenous metabolite of estradiol with no affinity for estrogen receptors, is a potent anticarcinogenic agent (in phase II clinical trials) and mediates the inhibitory effects of estradiol on smooth muscle cell (SMC) growth. Here we studied the intracellular mechanisms by which 2-ME inhibits SMC growth and whether 2-ME prevents injury-induced neointima formation. 2-ME concentrations that inhibit proliferation of cycling human aortic SMCs by >or=50% blocked cell-cycle progression in G(0)/G(1) and in G(2)/M phase, as determined by flow cytometry. Consistent with the cell-cycle effects, at a molecular level (Western blots), 2-ME inhibited cyclin D(1) and cyclin B(1) expression; cyclin-dependent kinase (cdk)-1 and cdk-2 activity; and retinoblastoma protein (pRb), extracellular signal-regulated kinase (ERK) 1/2, and Akt phosphorylation. 2-ME also upregulated the Cdk inhibitor p27 and interfered with tubulin polymerization. Moreover, 2-ME augmented COX-2 expression, suggesting that it may also inhibit SMC growth via prostaglandin formation. In rats, treatment with 2-ME abrogated injury-induced neointima formation; decreased proliferating SMCs; downregulated expression of proliferating-cell nuclear antigen (PCNA), c-myc, cyclin D(1), cyclin B(1), phosphorylated Akt, phosphorylated ERK1/2, p21, and pRb; inhibited cdk-1 and cdk-4 activity; and upregulated expression of cyclooxygenase (COX)-2 and p27. Caspase-3 cleavage assay and fluorescence-activated cell-sorting (FACS) analysis showed no evidence of apoptosis in 2-ME-treated SMCs, and TUNEL staining in carotid segments showed no evidence of 2-ME-induced apoptosis in vivo. The antimitotic effects of 2-ME on SMCs are mediated by the inhibition of key cell-cycle regulatory proteins and effects on tubulin polymerization and COX-2 upregulation. These effects of 2-ME most likely contribute to the antivasoocclusive actions of this endogenous compound.

Abstract-Estrogen receptors (ERs) are considered to mediate the ability of 17␤-estradiol (estradiol) to reduce injury-induced proliferation of vascular smooth muscle cells (VSMCs), leading to vascular lesions. However, the finding that estradiol attenuates formation of vascular lesions in response to vascular injury in knockout mice that lack either ER-␣ or ER-␤ challenges this concept. Our hypothesis is that the local metabolism of estradiol to methoxyestradiols, metabolites of estradiol with little affinity for ERs, mediates the ER-independent antimitogenic effects of estradiol on VSMCs. In human VSMCs, 2-methoxyestradiol and 2-hydroxyestradiol were more potent than was estradiol in inhibiting DNA synthesis ( 3 [H]-thymidine incorporation), collagen synthesis ( 3 [H]-proline incorporation), cell proliferation (cell number), and cell migration (movement of cells across a polycarbonate membrane). The inhibitory effects of estradiol on VSMCs were enhanced by cytochrome-P450 (CYP450) inducers 3-methylcholanthrene and phenobarbital. Moreover, the inhibitory effects of estradiol were blocked in the presence of the CYP450 inhibitor 1-aminobenzotriazole and the catechol-O-methyltransferase inhibitors quercetin and OR486. Both OR486 and quercetin blocked the conversion of 2-hydroxyestradiol to 2-methoxyestradiol; moreover, they blocked the antimitogenic effects of 2-hydroxyestradiol but not of 2-methoxyestradiol. The ER antagonist ICI182780 blocked the inhibitor effects of estradiol on VSMCs, but only at concentrations (Ͼ50 mol/L) that also inhibit the metabolism of estradiol to hydroxyestradiols (precursors of methoxyestradiols). In conclusion, the inhibitory effects of locally applied estradiol on human VSMCs are mediated via a novel ER-independent mechanism involving estradiol metabolism. These findings imply that vascular estradiol metabolism may be an important determinant of the cardiovascular protective effects of estradiol and that nonfeminizing estradiol metabolites may confer cardiovascular protection regardless of gender.

our data indicate that chymase inhibition can inhibit pro-atherogenic and plaque destabilizing effects which are associated with perivascular mast cell activation. Our study thus identifies pharmacological chymase inhibition as a potential therapeutic modality for atherosclerotic plaque stabilization.