Low dose oral cannabinoid therapy reduces progression of atherosclerosis in mice.

Atherosclerosis is a chronic inflammatory disease, and is the primary cause of heart disease and stroke in Western countries. Derivatives of cannabinoids such as delta-9-tetrahydrocannabinol (THC) modulate immune functions and therefore have potential for the treatment of inflammatory diseases. We investigated the effects of THC in a murine model of established atherosclerosis. Oral administration of THC (1 mg kg(-1) per day) resulted in significant inhibition of disease progression. This effective dose is lower than the dose usually associated with psychotropic effects of THC. Furthermore, we detected the CB2 receptor (the main cannabinoid receptor expressed on immune cells) in both human and mouse atherosclerotic plaques. Lymphoid cells isolated from THC-treated mice showed diminished proliferation capacity and decreased interferon-gamma secretion. Macrophage chemotaxis, which is a crucial step for the development of atherosclerosis, was also inhibited in vitro by THC. All these effects were completely blocked by a specific CB2 receptor antagonist. Our data demonstrate that oral treatment with a low dose of THC inhibits atherosclerosis progression in the apolipoprotein E knockout mouse model, through pleiotropic immunomodulatory effects on lymphoid and myeloid cells. Thus, THC or cannabinoids with activity at the CB2 receptor may be valuable targets for treating atherosclerosis.

Differential influence of chemokine receptors CCR2 and CXCR3 in development of atherosclerosis in vivo.

BACKGROUND: Recruitment of mononuclear leukocytes within atherosclerotic lesions is a critical step in atherogenesis. Mice lacking the chemokine receptor CCR2, highly expressed on macrophages but also on T lymphocytes, show a striking reduction of atherosclerotic lesion formation. The chemokine receptor CXCR3 is a marker of activated T helper type 1 lymphocytes, the principal T lymphocyte type detected within atheroma. We investigated whether the deletion of both of these 2 important receptors expressed on the principal inflammatory cells present in atheroma would further affect atherogenesis in vivo. METHODS AND RESULTS: We crossed ApoE(-/-) mice with either CCR2(-/-) or CXCR3- mice and crossed ApoE(-/-) CCR2(-/-) mice with the ApoE(-/-) CXCR3- mice to generate a triple knockout strain. Analysis of atherosclerosis development after 10 weeks of high-cholesterol diet revealed differential effects on early atherosclerotic lesions in the abdominal aorta and on advanced lesions in aortic roots. ApoE(-/-) CXCR3- mice, but not the triple knockout mice, displayed significantly reduced atherosclerotic lesion development within abdominal aortas compared with ApoE(-/-) CCR2(-/-) and ApoE(-/-) mice. This reduction of lesion formation correlated with an upregulation of antiinflammatory molecules such as interleukin-10, interleukin-18BP, and endothelial nitric oxide synthase and with an increased number of regulatory T lymphocytes within atherosclerotic lesions. In contrast, lesion size development within the aortic roots was more enhanced in ApoE(-/-) and ApoE(-/-) CXCR3- mice compared with ApoE(-/-) CCR2(-/-) and triple knockout mice. CONCLUSIONS: Blocking chemokine signaling in vivo through deletion of the chemokine receptors CCR2 and CXCR3 has differential effects during atherogenesis. In addition, our results point to an important role of regulatory T lymphocytes during early atherogenesis.

Simvastatin modulates chemokine and chemokine receptor expression by geranylgeranyl isoprenoid pathway in human endothelial cells and macrophages.

OBJECTIVE: Atherosclerosis is a chronic immuno-inflammatory disease involving the recruitment of monocytes and T lymphocytes to the vascular wall of arteries. Chemokines and their receptors, known to induce leukocyte migration, have recently been implicated in atherogenesis. Recent in vitro and in vivo studies have suggested that statins (3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors) have anti-inflammatory properties beyond their lipid-lowering effects. Thus, the aim of the present study was to investigate whether simvastatin reduces the expression of chemokines and chemokine receptors in two major cell types implicated in atherogenesis and to test isoprenoid intermediates involved in their regulation. METHODS AND RESULTS: We performed in vitro experiments on human vascular endothelial cells and human primary macrophages. First, we have shown by ELISA that 1 microM simvastatin significantly reduced MCP-1 in endothelial cells (ECs) and macrophages stimulated with TNF-alpha or IFN-gamma, respectively. Messenger RNA analysis revealed that expression of the chemokines MCP-1, MIP-1alpha and MIP-1beta, as well as the chemokine receptors CCR1, CCR2, CCR4 and CCR5, was decreased by simvastatin, both in ECs and macrophages. Furthermore, the statin effects were reversed by mevalonate and mimicked by the geranylgeranyl transferase inhibitor (GGTI), whereas the farnesyl transeferase inhibitor (FTI) had no effect. These results suggests that statins act via inhibition of the geranylgeranylation of proteins. CONCLUSIONS: Our results demonstrate that statins reduce chemokine and chemokine receptor expressions in human ECs and macrophages via inhibition of the geranylgeranylpyrophosphate pathway. Thus, our data provide further evidence that statins have anti-inflammatory properties beyond their lipid-lowering effects. These findings highlight specific novel therapeutic targets for cardiovascular diseases to reduce inflammation mediated by chemokines and their receptors.

Antagonism of RANTES receptors reduces atherosclerotic plaque formation in mice.

Increasing evidence supports the involvement of inflammation in the early phases of atherogenesis. Recruitment of leukocytes within the vascular wall, controlled by chemokines, is an essential process in the development of this common disease. In this study, we report that blocking a chemokine pathway in vivo with the CC chemokine antagonist Met-RANTES reduces the progression of atherosclerosis in a hypercholesterolemic mouse model. The reduction of lesions was correlated with a diminution of expression of several major chemokines and chemokine receptors, a decrease in leukocyte infiltration, and an increase of collagen-rich atheroma, features associated with stable atheroma. Treatment was well tolerated and serum lipid profiles were not affected. Whereas genetically engineered mice with deletion of either a CC chemokine or its receptor have demonstrated resistance to disease, to our knowledge, this is the first demonstration that treatment with a chemokine receptor antagonist limits the progression of atherosclerosis in vivo. Thus, our findings indicate that blockade of chemokine receptor/ligand interactions might become a novel therapeutic strategy to reduce the evolution of this common disease.

Statins reduce interleukin-6-induced C-reactive protein in human hepatocytes: new evidence for direct antiinflammatory effects of statins.

OBJECTIVE: Besides its predictive role in determining cardiovascular risk, C-reactive protein (CRP) may exert direct proatherogenic effects through proinflammatory properties. CRP is mainly produced by hepatocytes in response to interleukin-6 (IL-6) and is then released into the systemic circulation. 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors, or statins, significantly reduce cardiovascular events and mortality in patients with or without coronary artery disease and reduce plasma CRP levels in humans. However, the mechanism by which statins reduce plasma CRP levels remains unknown. METHODS AND RESULTS: In this study, we report that statins limit both protein and RNA levels of IL-6-induced CRP in human hepatocytes. These effects are reversed by l-mevalonate and mimicked by an inhibitor of the geranylgeranyltransferase. IL-6-induced CRP production requires the binding of IL-6 to its cognate receptors, which results in activation and phosphorylation of the transcription factor STAT3. We provide evidence that statins reduce this IL-6-induced phosphorylation of STAT3 in hepatocytes. CONCLUSIONS: These results demonstrate that statins reduce IL-6-induced CRP production directly in hepatocytes via inhibition of protein geranylgeranylation. We further show that statins act via inhibition of STAT3 phosphorylation. These findings furnish new evidence for direct antiinflammatory properties of statins and provide new mechanistic insight into their clinical benefits.

Cholesterol-independent effects of statins in inflammation, immunomodulation and atherosclerosis.

Atherosclerosis and its complications still represent the major cause of death in developed countries. Statins have revolutionized the treatment of dyslipidemia and demonstrated their ability to reduce and prevent coronary morbidity and mortality. Statins inhibit 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an enzyme crucial to cholesterol synthesis. The effectiveness and rapidity of statin-induced decreases in coronary events led to the speculation that statins possess cholesterol-independent effects. Since mevalonate produced by the HMG-CoA reductase is not only the precursor of cholesterol, but also of non steroidal isoprenoid compounds, such as the farnesyl pyrophosphate and the geranylgeranyl pyrophosphate, statins also regulate the small signaling proteins, Ras and Rho. Thus, inhibition of these prenylated proteins might account for the non-lipid lowering effects of statins. In this review, we describe the numerous beneficial pleiotropic effects of statins that could modulate atherogenesis.

Differential expression patterns of proinflammatory and antiinflammatory mediators during atherogenesis in mice.

OBJECTIVE: Recent advances support the current view of atherosclerosis as an inflammatory process that initiates and promotes lesion development to the point of acute thrombotic complications and clinical events. ApoE-deficient mice are a valuable model for studying the involvement of inflammatory mediators during atherogenesis. In this study, we investigated the correlation between atherosclerotic plaque development and expression of important pro- and antiinflammatory mediators during progression of atherosclerosis in ApoE-/- mice. METHODS AND RESULTS: Expression of proinflammatory cytokines, chemokines, and chemokine receptors within aortic lesions increased during atherogenesis, as detected by real-time quantitative reverse-transcription polymerase chain reaction. In parallel, the number of inflammatory cells within lesions increased together with serum cholesterol and body weight. Interestingly, the majority of inflammatory mediators investigated reached their maximum expression values at 10 weeks of diet, followed by continuous decrease of their expression levels, whereas atherosclerotic plaque size further increased. We show that the expression pattern of these different inflammatory mediators mainly correlates with the amount of inflammatory cells present within the atherosclerotic lesions. CONCLUSIONS: Atherosclerosis might result from an imbalance between pro- and antiinflammatory mediators in response to endothelial injury induced by cholesterol-rich diet. These data provide important information on the expression kinetics of inflammatory mediators and point out the possible role of antiinflammatory cells during atherogenesis.

Statins (HMG-CoA reductase inhibitors) reduce CD40 expression in human vascular cells.

OBJECTIVE: HMG-CoA reductase inhibitors (statins) possess anti-inflammatory and immunomodulatory properties that are independent of their lipid-lowering action. As the CD40-CD40L signaling pathway is implicated in the modulation of inflammatory responses between vascular cells, involving adhesion molecules, pro-inflammatory cytokines, chemokines, we sought to investigate the potential role of statins in regulating the expression of CD40. METHODS AND RESULTS: Using Western blot, flow cytometry and immunohistochemistry analyses, we observed that four different statins reduced IFN-gamma-induced CD40 expression in human vascular cells (endothelial cells, smooth muscle cells, macrophages and fibroblasts). This effect was dose-dependent (from 5 microM to 80 nM) and reversed by addition of L-mevalonate. Activation of vascular cells by human recombinant CD40L, as measured by ELISA for IL-6, IL-8 and MCP-1, was strongly reduced when cells were treated with statins. Immunostaining of human carotid atherosclerotic lesions of patients subjected to statin treatment revealed less CD40 expression on a 'per vascular cell' basis compared to control patients. Although many pleiotropic effects of statins are mediated by nitric oxide synthase (NOS)- or peroxisome proliferator-activated receptor (PPAR)-dependent signaling pathways, we observed similar statin-induced reduction of CD40 expression using NOS inhibitors or different PPAR ligands. CONCLUSION: Statins decrease CD40 expression and CD40-related activation of vascular cells. These effects are partially reversed by the HMG-CoA reductase product L-mevalonate and are mediated by NOS- or PPAR-dependent pathways. Altogether, these findings provide mechanistic insight into the beneficial effects of statins on atherogenesis. They also provide a scientific rationale for the use of statins as immunomodulators after organ transplantation.