Fabienne Burger

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-γ 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.

Ccr5 But Not Ccr1 Deficiency Reduces Development of Diet-Induced Atherosclerosis in Mice

Objective—Chemokines and their receptors are crucially involved in the development of atherosclerotic lesions by directing monocyte and T cell recruitment. The CC-chemokine receptors 1 (CCR1) and 5 (CCR5) expressed on these cells bind chemokines implicated in atherosclerosis, namely CCL5/RANTES. Although general blockade of CCL5 receptors reduces atherosclerosis, specific roles of CCR1 and CCR5 have not been unequivocally determined. Methods and Results—We provide two independent lines of investigation to dissect the effects of Ccr1 and Ccr5 deletion in apolipoprotein E–deficient (ApoE−/−) mice in a collaboration between Aachen/Germany and Geneva/Switzerland. Different strains of ApoE−/−Ccr5−/− mice, ApoE−/−Ccr1−/− mice or respective littermates, were fed a high-fat diet for 10 to 12 weeks. Plaque areas were quantified in the aortic roots and thoracoabdominal aortas. Concordantly, both laboratories found that lesion formation was reduced in ApoE−/−Ccr5−/− mice. Plaque quality and immune cells were assessed by immunohistochemistry or mRNA analysis. Whereas lesional macrophage content, aortic CD4, and Th1-related Tim3 expression were reduced, smooth muscle cell (SMC) content and expression of interleukin-10 in plaques, lesional SMCs, and splenocytes were elevated. Protection against lesion formation by Ccr5 deficiency was sustained over 22 weeks of high-fat diet or over 26 weeks of chow diet. Conversely, plaque area, T cell, and interferon-&ggr; content were increased in ApoE−/−Ccr1−/− mice. Conclusion—Genetic deletion of Ccr5 but not Ccr1 in ApoE−/− mice protects from diet-induced atherosclerosis, associated with a more stable plaque phenotype, reduced mononuclear cell infiltration, Th1-type immune responses, and increased interleukin-10 expression. This corroborates CCR5 as a promising therapeutic target.

Statins Reduce Interleukin-6–Induced C-Reactive Protein in Human Hepatocytes: New Evidence for Direct Antiinflammatory Effects of Statins

Objectives—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. Conclusion—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.

A Novel RANTES Antagonist Prevents Progression of Established Atherosclerotic Lesions in Mice

Background—Atherosclerosis is a chronic inflammatory disease that represents the primary cause of death through coronary disease and stroke. Chemokines are known to play a crucial role in this disease by recruiting inflammatory leukocytes to the endothelium. Recently, the chemokine variant [44AANA47]-RANTES was shown to impair inflammatory cell recruitment in vivo by interfering with heparin binding and oligomerization. Methods and Results—In this study we report that curative treatment with [44AANA47]-RANTES limits atherosclerotic plaque formation in LDLr−/− mice. This was associated with reduced infiltration of T cells and macrophages and reduced production of matrix metalloproteinase (MMP)-9. By contrast, the relative smooth muscle cell and collagen content was increased, indicating a more stable plaque phenotype. In addition, we provide evidence for direct inhibition of leukocyte recruitment into aortic root lesions, attenuated leukocyte rolling and arrest in mesenteric vessels, as well as a reduced proinflammatory response following Con A stimulation in vitro. Conclusions—Interference with chemokine oligomerization and chemokine/heparin interactions is a powerful novel approach that inhibits progression of established atherosclerosis in mice. By inhibiting leukocyte recruitment into plaques, [44AANA47]-RANTES mediates a less inflammatory plaque phenotype and thus reduced systemic inflammatory state.

CB2 cannabinoid receptor activation is cardioprotective in a mouse model of ischemia/reperfusion

Preventive treatment with cannabinoid agonists has been reported to reduce the infarct size in a mouse model of myocardial ischemia/reperfusion. Here we investigated the possible cardioprotective effect of selective CB(2) cannabinoid receptor activation during ischemia. We performed left coronary artery ligature in C57Bl/6 mice for 30 min, followed by 24 h of reperfusion. Five minutes before reperfusion, mice received intraperitoneal injection of the CB(2) selective agonist JWH-133 (20 mg/kg) or vehicle. Infarct size was assessed histologically and by cardiac troponin I (cTnI) ELISA. Immunohistochemical analysis of leukocyte infiltration, oxidative stress in situ quantification, real-time RT-PCR analysis of inflammatory mediators as well as western blots for kinase phosphorylation was also performed. In addition, we studied chemotaxis and integrin expression of human neutrophils in vitro. JWH-133 significantly reduced the infarct size (I/area at risk: 19.27%+/-1.91) as compared to vehicle-treated mice (31.77%+/-2.7). This was associated with a reduction of oxidative stress and neutrophil infiltration in the infarcted myocardium, whereas activation of ERK 1/2 and STAT-3 was increased. Preinjection of PI3K inhibitor LY294002, MEK 1/2 inhibitor U0126 and JAK-2 inhibitor AG-490 partially abrogated the JWH-133 mediated infarct size reduction. No changes in cardiac CXCL1, CXCL2, CCL3, TNF-alpha, and ICAM-1 expression levels were found. Furthermore, JWH-133 inhibited the TNF-alpha induced chemotaxis and integrin CD18/CD11b (Mac-1) upregulation on human neutrophils. Our data suggest that JWH-133 administration during ischemia reduces the infarct size in a mouse model of myocardial ischemia/reperfusion through a direct cardioprotective activity on cardiomyocytes and neutrophils.

HIV increases markers of cardiovascular risk: results from a randomized, treatment interruption trial.

Objectives:Plasma soluble inflammatory molecules are associated with the risk of ischaemic cardiovascular events. We investigated whether HIV replication modified the levels of these proteins in a combination antiretroviral therapy (cART) interruption trial. Method and results:In 145 HIV-infected Thai patients (62% women, median CD4 cell count 271 cells/μl, median plasma HIV-RNA 4.66 log10 copies/ml) included in the Swiss–Thai–Australia Treatment Interruption Trial (STACCATO) trial, leptin, adiponectin, C-reactive protein, soluble vascular cell adhesion molecule-1 (s-VCAM-1), P-selectin, chemokine ligand 2, chemokine ligand 3, interleukin (IL)-6, IL-10, granulocyte macrophage colony-stimulating factor and D-dimer were measured before cART was initiated, after cART had suppressed HIV replication to less than 50 copies/ml plasma (median 8 months) and again 12 weeks after randomization to continued cART (n = 48) or interrupted cART (n = 97). Multiple linear regression and logistic regression were used to investigate the association between each cardiovascular marker and plasma HIV-RNA. Initiation of cART resulted in significant declines in s-VCAM-1, P-selectin, leptin and D-dimer, whereas mediators with anti-inflammatory properties, such as adiponectin and IL-10, increased. At 12 weeks after randomization, we found positive associations between levels of s-VCAM-1 and chemokine ligand 2 with an increase in plasma HIV-RNA (r = 0.271, P = 0.001 and r = 0.24, P = 0.005, respectively), whereas levels of adiponectin decreased for each 1 log increase in plasma HIV-RNA (r = −0.24, P = 0.002). Detectable IL-10 was less likely (odds ratio = 0.64, 95% confidence interval = 0.43–0.96) for each 1 log increase in plasma HIV-RNA. Conclusion:Plasma levels of several inflammatory, anti-inflammatory and endothelial activation markers of cardiovascular disease are associated with HIV-RNA replication.

CB2 cannabinoid receptor agonist JWH-015 modulates human monocyte migration through defined intracellular signaling pathways.

Recruitment of leukocytes to inflammatory sites is crucial in the pathogenesis of chronic inflammatory diseases. The aim of this study was to investigate if activation of CB2 cannabinoid receptors would modulate the chemotactic response of human monocytes. Human monocytes treated with the CB2 agonist JWH-015 for 12-18 h showed significantly reduced migration to chemokines CCL2 and CCL3, associated with reduced mRNA and surface expression of their receptors CCR2 and CCR1. The induction of ICAM-1 in response to IFN-gamma was inhibited by JWH-015. Moreover, JWH-015 cross-desensitized human monocytes for migration in response to CCL2 and CCL3 by its own chemoattractant properties. The CB2-selective antagonist SR-144528, but not the CB1 antagonist SR-147778, reversed JWH-015-induced actions, whereas the CB2 agonist JWH-133 mimicked the effects of JWH-015. The investigation of underlying pathways revealed the involvement of phosphatidylinositol 3-kinase/Akt and ERK1/2 but not p38 MAPK. In conclusion, selective activation of CB2 receptors modulates chemotaxis of human monocytes, which might have crucial effects in chronic inflammatory disorders such as atherosclerosis or rheumatoid arthritis.

Short-Term Treatment With Anti-CD3 Antibody Reduces the Development and Progression of Atherosclerosis in Mice

Background— Atherosclerosis is a chronic inflammatory disease of the large arteries that is the primary cause of heart disease and stroke. Anti-CD3–specific antibodies suppress immune responses by antigenic modulation of the CD3 antibody/T-cell receptor complex. Their unique capacity to restore self-tolerance in a mouse model of diabetes and, importantly, in patients with recent-onset type 1 diabetes involves transforming growth factor-&bgr;–dependent mechanisms via expansion and/or activation of regulatory T cells. We hypothesized that treatment with anti-CD3–specific antibodies might inhibit atherosclerosis development and progression in mice. Methods and Results— Low-density lipoprotein receptor–deficient mice were fed a high-cholesterol diet for 13 or 24 weeks. Anti-CD3 antibody was administered on 5 consecutive days beginning 1 week before or 13 weeks after the high-cholesterol diet was initiated, respectively. Control mice were injected in parallel with phosphate-buffered saline. Anti-CD3 antibody therapy reduced plaque development when administered before a high-cholesterol diet and markedly decreased lesion progression in mice with already established atherosclerosis. We found increased production of the antiinflammatory cytokine transforming growth factor-&bgr; in concanavalin A–stimulated lymph node cells and enhanced expression of the regulatory T-cell marker Foxp3 in spleens of anti-CD3 antibody–treated mice. A higher percentage of apoptotic cells within the plaques of anti-CD3 antibody–treated mice was also observed. Conclusions— Altered disease progression, combined with the emergence of this particular cytokine pattern, indicates that short-term treatment with an anti-CD3 antibody induces a regulatory T-cell phenotype that restores self-tolerance in a mouse model of atherosclerosis.

Tumor necrosis factor-alpha (TNF-α) induces integrin CD11b/CD18 (Mac-1) up-regulation and migration to the CC chemokine CCL3 (MIP-1α) on human neutrophils through defined signalling pathways

Strong evidence suggests that neutrophils may play an active role in acute and chronic inflammatory disorders, such as rheumatoid arthritis and atherosclerosis. Given the role of pro-inflammatory cytokine TNF-alpha in these inflammatory processes, we planned the present study to investigate the effect of short term incubation with TNF-alpha on neutrophil migration to CCL3, a chemokine produced in inflammatory sites and normally devoid of neutrophil chemotactic properties. We found that TNF-alpha primed neutrophils for migration to CCL3 via CCR5. TNF-alpha-induced migration was a consequence of the TNF-alpha-induced up-regulation of integrin CD11b/CD18 (Mac-1) on neutrophil surface. Furthermore, TNF-alpha activity was found to be strictly dependent on the activation of ERK 1/2 p44, cooperating with the intracellular pathways involving Src kinases, PI3K/Akt, p38 MAPK, well known as activated in response to classical chemoattractants (CXCL8) or priming agents (GM-CSF). On the contrary, the effect of TNF-alpha on neutrophil migration to CCL3 was not dependent on JNK 1/2. In conclusion, the present report shows that TNF-alpha unveils a previously unknown capacity of neutrophils to migrate to CCL3 through the intervention of Mac-1. TNF-alpha regulates Mac-1 up-regulation through signalling pathways, involving various kinases, but not JNK 1/2. Although highly speculative, ERK 1/2 p44 may represent a selective target for the pharmacologic manipulation of neutrophil-mediated adverse activities in TNF-alpha-mediated inflammatory states.

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. Conclusion—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.