Graziano Pelli

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.

Antagonism of RANTES Receptors Reduces Atherosclerotic Plaque Formation in Mice

Abstract— 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.

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.

Reduced Connexin43 Expression Inhibits Atherosclerotic Lesion Formation in Low-Density Lipoprotein Receptor–Deficient Mice

Background—Gap junctions allow the direct exchange of ions and small molecules between cells in contact, thus coordinating physiological processes such as cell growth and differentiation. We have recently demonstrated increased expression of the gap junction protein connexin43 (Cx43) in specific subsets of cells in atherosclerotic lesions. Because the development of atherosclerosis depends critically on paracrine cell-to-cell interactions, we hypothesized that direct intercellular communication via gap junctions may be another factor controlling atherogenesis. Methods and Results—The role of Cx43 in atherogenesis was examined by use of both a genetic and a pharmacological approach. First, atherosclerosis-susceptible LDL receptor–deficient (LDLR−/−) mice with normal (Cx43+/+) or reduced (Cx43+/−) levels of Cx43 were fed a cholesterol-rich diet for 14 weeks. The progression of atherosclerosis was reduced by 50% (P <0.01) in the thoracoabdominal aorta and in the aortic roots of Cx43+/−LDLR−/− mice compared with Cx43+/+LDLR−/− controls. Atheroma in Cx43+/−LDLR−/− mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells. Next, we observed that HMG-CoA reductase inhibitors, or “statins,” lipid-lowering drugs well known for their pleiotropic antiatherogenic effects, reduced Cx43 expression in primary human vascular cells in vitro. Atheroma of LDLR−/− mice treated orally with pravastatin contained fewer inflammatory cells and exhibited thicker fibrous caps than controls. This was associated with reduced Cx43 expression in lesions of statin-treated mice. Conclusions—These data indicate a critical role for Cx43-mediated gap junctional communication in atherosclerotic plaque formation.

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.

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.

Anti-Apolipoprotein A-1 auto-antibodies are active mediators of atherosclerotic plaque vulnerability

AIMS Anti-Apolipoprotein A-1 auto-antibodies (anti-ApoA-1 IgG) represent an emerging prognostic cardiovascular marker in patients with myocardial infarction or autoimmune diseases associated with high cardiovascular risk. The potential relationship between anti-ApoA-1 IgG and plaque vulnerability remains elusive. Thus, we aimed to investigate the role of anti-ApoA-1 IgG in plaque vulnerability. METHODS AND RESULTS Potential relationship between anti-ApoA-1 IgG and features of cardiovascular vulnerability was explored both in vivo and in vitro. In vivo, we investigated anti-ApoA-1 IgG in patients with severe carotid stenosis (n = 102) and in ApoE-/- mice infused with polyclonal anti-ApoA-1 IgG. In vitro, anti-ApoA-1 IgG effects were assessed on human primary macrophages, monocytes, and neutrophils. Intraplaque collagen was decreased, while neutrophil and matrix metalloprotease (MMP)-9 content were increased in anti-ApoA-1 IgG-positive patients and anti-ApoA-1 IgG-treated mice when compared with corresponding controls. In mouse aortic roots (but not in abdominal aortas), treatment with anti-ApoA-1 IgG was associated with increased lesion size when compared with controls. In humans, serum anti-ApoA-1 IgG levels positively correlated with intraplaque macrophage, neutrophil, and MMP-9 content, and inversely with collagen. In vitro, anti-ApoA-1 IgG increased macrophage release of CCL2, CXCL8, and MMP-9, as well as neutrophil migration towards TNF-α or CXCL8. CONCLUSION These results suggest that anti-ApoA-1 IgG might be associated with increased atherosclerotic plaque vulnerability in humans and mice.

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.

Reduced Connexin43 Expression Limits Neointima Formation After Balloon Distension Injury in Hypercholesterolemic Mice

Background— Reducing the expression of the gap junction protein connexin43 (Cx43) inhibits the progression of atherosclerosis, a chronic inflammatory disease. Furthermore, acute vascular injury induced by percutaneous coronary interventions is associated with increased Cx43 expression in neointimal smooth muscle cells (SMCs). However, the relevance of Cx43 after acute vascular injury remains unclear. Methods and Results— To investigate whether reducing Cx43 expression would affect neointima formation in vivo, we subjected hypercholesterolemic Cx43+/− LDL receptor–deficient (LDLR−/−) mice and Cx43+/+LDLR−/− control littermates to carotid balloon distension injury, which induced marked endothelial denudation and activation of medial SMCs. We observed decreased macrophage infiltration in Cx43+/−LDLR−/− mice 7 days after injury. Similarly, peritoneal macrophages isolated from Cx43+/−LDLR−/− mice showed reduced migration in vitro compared with Cx43+/+LDLR−/− macrophages. Interestingly, Cx43+/−LDLR−/− macrophages also displayed decreased chemotactic activity for SMCs. In addition, we observed less SMC infiltration and proliferation in Cx43+/−LDLR−/− mice 7 and 14 days after balloon angioplasty. Likewise, Cx43+/−LDLR−/− SMCs showed decreased proliferation and migration in vitro compared with Cx43+/+LDLR−/− cells. All these events resulted in a reduction of neointimal thickening after vascular injury in Cx43+/−LDLR−/− mice. Conclusions— The present study shows for the first time that reducing Cx43 limits neointima formation after acute vascular injury by decreasing the inflammatory response and reducing SMC migration and proliferation. Thus, decreasing Cx43 expression may offer a novel therapeutic strategy for reducing restenosis after percutaneous coronary intervention.