Pharmacological Treatment with Annexin A1 Reduces Atherosclerotic Plaque Burden in LDLR-/- Mice on Western Type Diet.
ABSTRACT: To investigate therapeutic effects of annexin A1 (anxA1) on atherogenesis in LDLR-/- mice.Human recombinant annexin A1 (hr-anxA1) was produced by a prokaryotic expression system, purified and analysed on phosphatidylserine (PS) binding and formyl peptide receptor (FPR) activation. Biodistribution of 99mTechnetium-hr-anxA1 was determined in C57Bl/6J mice. 12 Weeks old LDLR-/- mice were fed a Western Type Diet (WTD) during 6 weeks (Group I) or 12 weeks (Group P). Mice received hr-anxA1 (1 mg/kg) or vehicle by intraperitoneal injection 3 times per week for a period of 6 weeks starting at start of WTD (Group I) or 6 weeks after start of WTD (Group P). Total aortic plaque burden and phenotype were analyzed using immunohistochemistry.Hr-anxA1 bound PS in Ca2+-dependent manner and activated FPR2/ALX. It inhibited rolling and adherence of neutrophils but not monocytes on activated endothelial cells. Half lives of circulating 99mTc-hr-anxA1 were <10 minutes and approximately 6 hours for intravenously (IV) and intraperitoneally (IP) administered hr-anxA1, respectively. Pharmacological treatment with hr-anxA1 had no significant effect on initiation of plaque formation (-33%; P = 0.21)(Group I) but significantly attenuated progression of existing plaques of aortic arch and subclavian artery (plaque size -50%, P = 0.005; necrotic core size -76% P = 0.015, hr-anxA1 vs vehicle) (Group P).Hr-anxA1 may offer pharmacological means to treat chronic atherogenesis by reducing FPR-2 dependent neutrophil rolling and adhesion to activated endothelial cells and by reducing total plaque inflammation.
Project description:This study focused on the unique properties of both the Ldlr knockout defect (closely mimicking the human situation) and the BALB/c (C) inbred mouse strain (Th-2 slanted immune response). We generated two immunodeficient strains with severe combined B- and T-cell immunodeficiency with or without a complete lack of natural killer cells to revisit the role of adaptive immune responses on atherogenesis. C-Ldlr-/- Rag1-/- mice, which show severe combined B- and T-cell immunodeficiency and C-Ldlr-/- Rag1-/- Il2rg-/- mice, which combine the T- and B-cell defect with a complete lack of natural killer cells and inactivation of multiple cytokine signalling pathways were fed an atherogenic Western type diet (WTD). Both B6-Ldlr-/- and C-Ldlr-/- immunocompetent mice were used as controls. Body weights and serum cholesterol levels of both immunodeficient strains were significantly increased compared to C-Ldlr-/- controls, except for cholesterol levels of C-Ldlr-/- Rag1-/- double mutants after 12 weeks on the WTD. Quantification of the aortic sinus plaque area revealed that both strains of immunodeficient mice developed significantly more atherosclerosis compared to C-Ldlr-/- controls after 24 weeks on the WTD. Increased atherosclerotic lesion development in C-Ldlr-/- Rag1-/- Il2rg-/- triple mutants was associated with significantly increased numbers of macrophages and significantly decreased numbers of smooth muscle cells compared to both C-Ldlr-/- wild type and C-Ldlr-/- Rag1-/- double mutants pointing to a plaque destabilizing effect of NK cell loss. Collectively, the present study reveals a previously unappreciated complexity with regard to the impact of lymphocytes on lipoprotein metabolism and the role of lymphocyte subsets in plaque composition.
Project description:Although macrophages represent the hallmark of both human and murine atherosclerotic lesions and have been shown to express TGF-ß1 (transforming growth factor β1) and its receptors, it has so far not been experimentally addressed whether the pleiotropic cytokine TGF-ß1 may influence atherogenesis by a macrophage specific mechanism. We developed transgenic mice with macrophage specific TGF-ß1 overexpression, crossed the transgenics to the atherosclerotic ApoE (apolipoprotein E) knock-out strain and quantitatively analyzed both atherosclerotic lesion development and composition of the resulting double mutants. Compared with control ApoE(-/-) mice, animals with macrophage specific TGF-ß1 overexpression developed significantly less atherosclerosis after 24 weeks on the WTD (Western type diet) as indicated by aortic plaque area en face (p<0.05). Reduced atherosclerotic lesion development was associated with significantly less macrophages (p<0.05 after both 8 and 24 weeks on the WTD), significantly more smooth muscle cells (SMCs; p<0.01 after 24 weeks on the WTD), significantly more collagen (p<0.01 and p<0.05 after 16 and 24 weeks on the WTD, respectively) without significant differences of inner aortic arch intima thickness or the number of total macrophages in the mice pointing to a plaque stabilizing effect of macrophage-specific TGF-ß1 overexpression. Our data shows that macrophage specific TGF-ß1 overexpression reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice.
Project description: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.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.CB2 receptor 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.
Project description:LDL-cholesterol (LDL-C) is a causal pathogenic factor in atherosclerosis. Monoclonal anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) neutralizing antibodies are novel potent LDL-lowering drugs which reduce cardiovascular events. To characterize their effect on atherogenesis, APOE*3Leiden.CETP mice were fed a high cholesterol/high fat diet (WTD) or normal chow (NC) for 18 weeks. Mice on WTD were injected with the human anti-PCSK9 antibody mAb1 (PL-45134, 10?mg*kg-1 s.c.) or 0.9% saline every 10 days. PCSK9 inhibition decreased total cholesterol in serum of APOE*3Leiden.CETP mice and prevented the development of atherosclerosis. The plaque area in the aortic root was reduced by half and macrophage infiltration determined by Ly6c and Mac-3 staining was ameliorated. PCSK9 inhibition decreased markers of inflammation in mononuclear cells (Il-6, Tnfa mRNA), and in serum (CXCL-1,-10,-13; complement factor C5a) compared to control WTD fed animals. The number of circulating Sca-1/VEGF-R2 positive endothelial progenitor cells of the peripheral blood and spleen-derived diLDL/lectin double positive circulating angiogenic cells was increased. To conclude, the PCSK9-mediated anti-atherosclerotic effect involves the upregulation of pro-regeneratory endothelial progenitor cells, a reduction of inflammation and change of plaque composition.
Project description:Proapoptotic Bcl-2 family member Bim is particularly relevant for deletion of autoreactive and activated T and B cells, implicating Bim in autoimmunity. As atherosclerosis is a chronic inflammatory process with features of autoimmune disease, we investigated the impact of hematopoietic Bim deficiency on plaque formation and parameters of plaque stability. Bim -/- or wild type bone marrow transplanted ldlr -/- mice were fed a Western type diet (WTD) for 5 or 10 weeks, after which they were immunophenotyped and atherosclerotic lesions were analyzed. Bim -/- transplanted mice displayed splenomegaly and overt lymphocytosis. CD4+ and CD8+ T cells were more activated (increased CD69 and CD71 expression, increased interferon gamma production). B cells were elevated by 147%, with a shift towards the pro-atherogenic IgG-producing B2 cell phenotype, resulting in a doubling of anti-oxLDL IgG1 antibody titers in serum of bim -/- mice. Bim -/- mice displayed massive intraplaque accumulation of Ig complexes and of lesional T cells, although this did not translate in changes in plaque size or stability features (apoptotic cell and macrophage content). The surprising lack in plaque phenotype despite the profound pro-atherogenic immune effects may be attributable to the sharp reduction of serum cholesterol levels in WTD fed bim -/- mice.
Project description:The N-formyl peptide receptors (FPRs) are a family of G-protein coupled receptors that respond to proinflammatory N-formylated bacterial peptides (e.g., formyl-Met-Leu-Phe, fMLF) and, thus, contribute to the host response to bacterial infection. Paradoxically, a growing body of evidence suggests that some members of this receptor family may also be targets for certain anti-inflammatory molecules, including annexin A1 (ANXA1), which is an important mediator of glucocorticoid (GC) action. To explore further the potential role of FPRs in mediating ANXA1 actions, we have focused on the pituitary gland, where ANXA1 has a well-defined role as a cell-cell mediator of the inhibitory effects of GCs on the secretion of corticotrophin (ACTH), and used molecular, genetic, and pharmacological approaches to address the question in well-established rodent models. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis identified mRNAs for four FPR family members in the mouse anterior pituitary gland, Fpr-rs1, Fpr-rs2, Fpr-rs6, and Fpr-rs7. Functional studies confirmed that, like dexamethasone, ANXA1 and two ANXA1-derived peptides (ANXA1(1-188) and ANXA1(Ac2-26)) inhibit the evoked release of ACTH from rodent anterior pituitary tissue in vitro. Fpr1 gene deletion failed to modify the pituitary responses to dexamethasone or ANXA1(Ac2-26). However, lipoxin A4 (LXA4, 0.02-2 microM, a lipid mediator with high affinity for Fpr-rs1) mimicked the inhibitory effects of ANXA1 on ACTH release as also did fMLF in high (1-100 microM) but not lower (10-100 nM) concentrations. Additionally, a nonselective FPR antagonist (Boc1, 100 microM) overcame the effects of dexamethasone, ANXA1(1-188), ANXA1(Ac2-26), fMLF, and LXA4 on ACTH release, although at a lower concentration (50 microM), it was without effect. Together, the results suggest that the actions of ANXA1 in the pituitary gland are independent of Fpr1 but may involve other FPR family members, in particular, Fpr-rs1 or a closely related receptor. They thus provide the first evidence for a role of the FPR family in the regulation of neuroendocrine function.
Project description:BACKGROUND AND PURPOSE:Atherosclerosis results from a maladaptive inflammatory response initiated by the intramural retention of LDL in susceptible areas of the arterial vasculature. The ω-3 polyunsaturated fatty acids (ω-3) have protective effects in atherosclerosis; however, their molecular mechanism is still largely unknown. The present study used a metabolomic approach to reveal the atheroprotective metabolites of ω-3 and investigate the underlying mechanisms. EXPERIMENTAL APPROACH:We evaluated the development of atherosclerosis in LDL receptor-deficient mice (LDLR-/- ) fed a Western-type diet (WTD) plus ω-3 and also LDLR-/- and fat-1 transgenic (LDLR-/- -fat-1tg ) mice fed a WTD. The profiles of ω-3 in the plasma were screened by LC-MS/MS using unbiased systematic metabolomics analysis. We also studied the effect of metabolites of eicosapentaenoic acid (EPA) on endothelial activation in vitro. KEY RESULTS:The ω-3 diet and fat-1 transgene decreased monocyte infiltration, inhibited the expression of pro-inflammatory genes and significantly attenuated atherosclerotic plaque formation and enhanced plaque stability in LDLR-/- mice. The content of 18-hydroxy-eicosapentaenoic acid (18-HEPE) and 17,18-epoxy-eicosatetraenoic acid (17,18-EEQ), from the cytochrome P450 pathway of EPA, was significantly higher in plasma from both ω-3-treated LDLR-/- and LDLR-/- -fat-1tg mice as compared with WTD-fed LDLR-/- mice. In vitro in endothelial cells, 18-HEPE or 17,18-EEQ decreased inflammatory gene expression induced by TNFα via NF-κB signalling and thereby inhibited monocyte adhesion to endothelial cells. CONCLUSIONS AND IMPLICATIONS:EPA protected against the development of atherosclerosis in atheroprone mice via the metabolites 18-HEPE and/or 17,18-EEQ, which reduced endothelial activation. These compounds may have therapeutic implications in atherosclerosis. LINKED ARTICLES:This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.
Project description:BACKGROUND AND AIMS:Altered metabolism is an important regulator of macrophage (M?) phenotype, which contributes to inflammatory diseases such as atherosclerosis. Broadly, pro-inflammatory, classically-activated M?s (CAM) are glycolytic while alternatively-activated M?s (AAM) oxidize fatty acids, although overlap exists. We previously demonstrated that M? fatty acid transport protein 1 (FATP1, Slc27a1) was necessary to maintain the oxidative and anti-inflammatory AAM phenotype in vivo in a model of diet-induced obesity. The aim of this study was to examine how M? metabolic reprogramming through FATP1 ablation affects the process of atherogenesis. We hypothesized that FATP1 limits M?-mediated inflammation during atherogenesis. Thus, mice lacking M? Fatp1 would display elevated formation of atherosclerotic lesions in a mouse model lacking the low-density lipoprotein (LDL) receptor (Ldlr-/-). METHODS:We transplanted bone marrow collected from Fatp1+/+ or Fatp1-/- mice into Ldlr-/- mice and fed chimeric mice a Western diet for 12 weeks. Body weight, blood glucose, and plasma lipids were measured. Aortic sinus and aorta lesions were quantified. Atherosclerotic plaque composition, oxidative stress, and inflammation were analyzed histologically. RESULTS:Compared to Fatp1+/+Ldlr-/- mice, Fatp1-/-Ldlr-/- mice exhibited significantly larger lesion area and elevated oxidative stress and inflammation in the atherosclerotic plaque. Macrophage and smooth muscle cell content did not differ by Fatp1 genotype. There were no significant systemic alterations in LDL, high-density lipoprotein (HDL), total cholesterol, or triacylglyceride, suggesting that the effect was local to the cells of the vessel microenvironment in a Fatp1-dependent manner. CONCLUSIONS:M? Fatp1 limits atherogenesis and may be a viable target to metabolically reprogram M?s.
Project description:Non-alcoholic fatty liver disease is a spectrum of liver diseases ranging from steatosis only to non-alcoholic steatohepatitis (NASH). The latter is characterized by hepatic inflammation, which increases the risk of cardiovascular disease. It is poorly understood which factors contribute to the onset of hepatic inflammation characterizing the progression from steatosis to NASH. Previously, we demonstrated increased advanced glycation endproducts (AGEs) in the livers of NASH patients. We hypothesise that AGEs play a key role in NASH development by activating their proinflammatory receptor, RAGE. RAGE-deficient mice and wildtype littermates, both on Ldlr-/- background, were fed a Western type diet (WTD) for 3 or 12 weeks. Flow cytometry, histology, gene expression and AGE measurements were performed to evaluate the effects of RAGE deficiency. RAGE-deficient mice displayed reduced weight gain and visceral fat expansion compared to control mice. No difference in adipose tissue inflammation was observed between groups. RAGE deficiency did not affect WTD-induced monocytosis, circulating lipids or hepatic steatosis. WTD-induced hepatic neutrophil and macrophage accumulation and atherosclerotic plaque development was comparable between control and RAGE-deficient mice. No difference in AGE levels was observed. RAGE does not seem to play a major role in the development of NASH or atherosclerosis in a hyperlipidemic mouse model.
Project description:Blood-brain barrier (BBB) disruption is associated with hypoxia-ischemia (HI) induced brain injury and life-long neurological pathologies. Treatment options are limited. Recently, we found that mesenchymal stem/stromal cell derived extracellular vesicles (MSC-EVs) protected the brain in ovine fetuses exposed to HI. We hypothesized that Annexin A1 (ANXA1), present in MSC-EVs, contributed to their therapeutic potential by targeting the ANXA1/Formyl peptide receptor (FPR), thereby preventing loss of the BBB integrity. Cerebral ANXA1 expression and leakage of albumin into the fetal ovine brain parenchyma after HI were analyzed by immunohistochemistry. For mechanistic insights, barrier integrity of primary fetal endothelial cells was assessed after oxygen-glucose deprivation (OGD) followed by treatment with MSC-EVs or human recombinant ANXA1 in the presence or absence of FPR inhibitors. Our study revealed that BBB integrity was compromised after HI which was improved by MSC-EVs containing ANXA1. Treatment with these MSC-EVs or ANXA1 improved BBB integrity after OGD, an effect abolished by FPR inhibitors. Furthermore, endogenous ANXA1 was depleted within 24 h after induction of HI in cerebovasculature and ependyma and upregulated 72 h after HI in microglia. Targeting ANXA1/FPR with ANXA1 in the immature brain has great potential in preventing BBB loss and concomitant brain injury following HI.