Plaques In ApoE Knockout Mice Research Articles

The role of macrophage RIPK1 in the development of atherosclerotic plaques in APOE knockout mice

Background and Aims: During atherogenesis, macrophages undergo necrosis and release their cytoplasmic content in the plaque, thereby forming a necrotic core. Further expansion of the necrotic core promotes plaque instability and rupture. Hence, targeting macrophage death is a promising strategy to stabilize plaques. Recently, necroptosis was discovered as a form of regulated necrosis. The formation of a receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 pro-necrotic complex is crucial for necroptosis induction. This study aimed to investigate the impact of a macrophage-specific RIPK1 deletion on atherogenesis.

Effects of nanoparticle-mediated delivery of pitavastatin on atherosclerotic plaques in ApoE-knockout mice and THP-1-derived macrophages.

The treatment of atherosclerosis remains complex. Pitavastatin serves an important role in the prevention and treatment of atherosclerosis. The present study aimed to investigate the effects of nanoparticle (NP)-mediated delivery of pitavastatin into atherosclerotic plaques as a novel treatment method for atherosclerosis. The results of the present study demonstrated that pitavastatin-NP was more effective in attenuating the size of atherosclerotic plaques and enhancing the stability of plaques in vitro compared with pitavastatin alone. In an apolipoprotein E (ApoE)-knockout mouse model of atherosclerosis, a single intravenous injection of fluorescein isothiocyanate-NP resulted in the delivery of NP into atherosclerotic plaques for up to 7 days post-injection. In ApoE-knockout mice and THP-1-derived macrophages, pitavastatin-NP attenuated the development of atherosclerosis, which was associated with regulating lipid metabolism, and inhibited the secretion of inflammatory markers compared with pitavastatin alone. Additionally, the treatment advantages of pitavastatin-NP were independent of lipid lowering. The results demonstrated that pitavastatin-NP administration was more effective in attenuating the development of atherosclerotic plaques compared with systemic administration of pitavastatin.

Tanshinone IIA harmonizes the crosstalk of autophagy and polarization in macrophages via miR-375/KLF4 pathway to attenuate atherosclerosis

Macrophages play a pivotal role in destabilizing atherosclerotic plaque. The diverse phenotypes and complex autophagy in macrophage are observed in atherosclerotic lesions. Tanshinone IIA (TNA) is known as the major component extracted from the root of Chinese herb Salvia miltiorrhiza, used for treatment of cardiovascular diseases. However, the therapeutic mechanism of TNA is not clear yet. In this study, we identified inflammation-related gene expression by microarray in atherosclerotic plaques in ApoE knockout mice fed with high fat diet and found miR-375 was one of the significantly high expressed microRNAs compared with wild type mice and TNA treated mice. Then we compared the levels of proteins related to the signal pathway of autophagy, and the phenotype of macrophages in atherosclerotic plaques ex vivo. We predicted KLF4 might be the key target of miR-375 that mediated the crosstalk between autophagy and polarization by TNA. Furthermore, we detected the expression of signal pathway in ox-LDL induced macrophages after treatment with TNA in vitro to verify this predict. The results suggest TNA could activate KLF4 and enhance autophagy as well as M2 polarization of macrophages by inhibiting miR-375 to Attenuate Atherosclerosis.

Macrophage-specific RIP1 deletion reduces necrotic core formation in atherosclerotic plaques of APOE knockout mice

Aim: During atherogenesis, macrophages undergo necrosis and release their cytoplasmic content in the centre of the plaque, thereby forming a necrotic core. Further expansion of the necrotic core promotes plaque instability and rupture. Hence, targeting macrophage death is a promising strategy to stabilize atherosclerotic plaques. Recently, necroptosis was discovered as a form of regulated necrosis. The formation of a RIP1 and RIP3 pro-necrotic complex, called the necrosome, is a crucial step in the induction of necroptosis. This study aimed to investigate the impact of a macrophage specific RIP1 deletion on atherogenesis.

Antiinflammatory actions of inorganic nitrate stabilize the atherosclerotic plaque

Reduced bioavailable nitric oxide (NO) plays a key role in the enhanced leukocyte recruitment reflective of systemic inflammation thought to precede and underlie atherosclerotic plaque formation and instability. Recent evidence demonstrates that inorganic nitrate (NO3-) through sequential chemical reduction in vivo provides a source of NO that exerts beneficial effects upon the cardiovascular system, including reductions in inflammatory responses. We tested whether the antiinflammatory effects of inorganic nitrate might prove useful in ameliorating atherosclerotic disease in Apolipoprotein (Apo)E knockout (KO) mice. We show that dietary nitrate treatment, although having no effect upon total plaque area, caused a reduction in macrophage accumulation and an elevation in smooth muscle accumulation within atherosclerotic plaques of ApoE KO mice, suggesting plaque stabilization. We also show that in nitrate-fed mice there is reduced systemic leukocyte rolling and adherence, circulating neutrophil numbers, neutrophil CD11b expression, and myeloperoxidase activity compared with wild-type littermates. Moreover, we show in both the ApoE KO mice and using an acute model of inflammation that this effect upon neutrophils results in consequent reductions in inflammatory monocyte expression that is associated with elevations of the antiinflammatory cytokine interleukin (IL)-10. In summary, we demonstrate that inorganic nitrate suppresses acute and chronic inflammation by targeting neutrophil recruitment and that this effect, at least in part, results in consequent reductions in the inflammatory status of atheromatous plaque, and suggest that this effect may have clinical utility in the prophylaxis of inflammatory atherosclerotic disease.

Caspase-3 Deletion Promotes Necrosis in Atherosclerotic Plaques of ApoE Knockout Mice.

Apoptosis of macrophages and vascular smooth muscle cells (VSMCs) in advanced atherosclerotic plaques contributes to plaque progression and instability. Caspase-3, a key executioner protease in the apoptotic pathway, has been identified in human and mouse atherosclerotic plaques but its role in atherogenesis is not fully explored. We therefore investigated the impact of caspase-3 deletion on atherosclerosis by crossbreeding caspase-3 knockout (Casp3−/−) mice with apolipoprotein E knockout (ApoE−/−) mice. Bone marrow-derived macrophages and VSMCs isolated from Casp3−/−ApoE−/− mice were resistant to apoptosis but showed increased susceptibility to necrosis. However, caspase-3 deficiency did not sensitize cells to undergo RIP1-dependent necroptosis. To study the effect on atherosclerotic plaque development, Casp3+/+ApoE−/− and Casp3−/−ApoE−/− mice were fed a western-type diet for 16 weeks. Though total plasma cholesterol, triglycerides, and LDL cholesterol levels were not altered, both the plaque size and percentage necrosis were significantly increased in the aortic root of Casp3−/−ApoE−/− mice as compared to Casp3+/+ApoE−/− mice. Macrophage content was significantly decreased in plaques of Casp3−/−ApoE−/− mice as compared to controls, while collagen content and VSMC content were not changed. To conclude, deletion of caspase-3 promotes plaque growth and plaque necrosis in ApoE−/− mice, indicating that this antiapoptotic strategy is unfavorable to improve atherosclerotic plaque stability.

Anti eotaxin-2 antibodies attenuate the initiation and progression of experimental atherosclerosis

Background: The chemokine eotaxin-2 is a potent chemoattractant for inflammatory cells, the predominants of which are eosinophils. Human and murine atherosclerotic plaques are known to exhibit inflammatory phenotypes where a complex interaction of cytokine and chemokines plays a role. We tested the hypothesis that eotaxin-2 (eo-2) plays a causative role in the initiation and progression of experimental atherosclerosis. Methods and Results: Sera collected from atherosclerotic ApoE knockout (KO) mice, exhibited significantly higher levels of eo-2 compared to sera collected from their background age matched C57BL/6 litters by ELISA. Moreover, levels of eo-2 were higher in old atherosclerotic ApoE KO mice than in young animals. Similarly, the expression level of the eo-2 receptor, CCR3, was increased in splenocytes of old ApoE compared to the young littermates. Administration of polyclonal blocking antibodies to eotaxin-2 resulted in a significant reduction of early atherosclerotic plaques in ApoE KO mice whereas prolonged treatment of mice with advanced plaques led to atheroma stabilization. A monoclonal antibody (D8) prepared against eo-2 attenuated adhesion of lymphocytes to fibronectin and potently inhibited their migration towards VEGF. Monoclonal blocking antibodies to eo-2 also significantly reduced atherosclerotic plaques in ApoE KO mice. Conclusion: Eo-2 serum levels are elevated in sera of ApoE KO mice with experimental atherosclerosis and its blockade is associated with reduced fatty streak accumulation and increased plaque stabilization.

Angiotensin II induces extracellular matrix metalloproteinase inducer expression via an AT1R dependent pathway in aortic atherosclerotic plaque in apolipoprotein E knockout mice

The pathogenesis of acute coronary syndrome is rupture of vulnerable plaque. Extracellular matrix metalloproteinase inducer (EMMPRIN) is reported to have a important role in the destabilization of atheroma. this investigation examined the effect of angiotensin II (Ang II) on EMMPRIN expression in atherosclerotic plaques in ApoE knockout mice. ApoE knockout mice were fed a high fat diet to establish an atherosclerosis model then intervention was made with Ang II and valsartan. EMMPRIN gene and its protein expression were measured by real-time PCR immunohistochemistry, and Western blot. EMMPRIN gene and protein expression intervened with Ang II were significantly increased; valsartan could inhibit the effect of Ang II. Ang II up-regulated EMMPRIN expression in atherosclerotic plaque via AT1R, and valsartan could inhibit the effect of Ang II.

Macrophage Plasticity in Experimental Atherosclerosis

As in human disease, macrophages (MØ) are central players in the development and progression of experimental atherosclerosis. In this study we have evaluated the phenotype of MØ associated with progression of atherosclerosis in the apolipoprotein E (ApoE) knockout (KO) mouse model.We found that bone marrow-derived MØ submitted to M1 and M2 polarization specifically expressed arginase (Arg) II and Arg I, respectively. This distinct arginase expression was used to evaluate the frequency and distribution of M1 and M2 MØ in cross-sections of atherosclerotic plaques of ApoE KO mice. Early lesions were infiltrated by Arg I+ (M2) MØ. This type of MØ favored the proliferation of smooth muscle cells, in vitro. Arg II+ (M1) MØ appeared and prevailed in lesions of aged ApoE KO mice and lesion progression was correlated with the dominance of M1 over the M2 MØ phenotype. In order to address whether the M2->M1 switch could be due to a phenotypic switch of the infiltrated cells, we performed in vitro repolarization experiments. We found that fully polarized MØ retained their plasticity since they could revert their phenotype. The analysis of the distribution of Arg I- and Arg II-expressing MØ also argued against a recent recruitment of M1 MØ in the lesion. The combined data therefore suggest that the M2->M1 switch observed in vivo is due to a conversion of cells already present in the lesion. Our study suggests that interventional tools able to revert the MØ infiltrate towards the M2 phenotype may exert an atheroprotective action.

Expression of neuronal nitric oxide synthase splice variants in atherosclerotic plaques of apoE knockout mice

Objective We previously reported that deletion of brain type neuronal nitric oxide synthase-α (nNOS-α) accelerates atherosclerosis in apolipoproteinE (apoE) knockout (ko) mice. The regulation of nNOS expression is complex, involving the generation of mRNA splice variants. The current study investigates occurrence and distribution of nNOS variants in atherosclerotic lesions of apoE ko and apoE/nNOS-α double ko (dko) animals. Methods Mice were fed a high fat diet for 20 weeks. Immunohistochemistry and western blot analysis were performed using antibodies detecting the carboxy terminal-, or amino terminal-residue of the nNOS protein. Confocal microscopy and in situ hybridization were used to identify the compartment of cellular expression. Results In situ hybridization revealed the presence of nNOS-α and -γ mRNA variants in apoE ko plaques, while only nNOS-γ was detectable in apoE/nNOS dko plaques. Consistent with mRNA expression nNOS-α protein can be detected in the neointima of apoE ko, but not apoE/nNOS dko animals. In contrast, the carboxy terminal antibody stained the neointima and media in apoE ko vessels and showed residual nNOS immunoreactivity in apoE/nNOS dko lesions. Confocal microscopy showed predominant nNOS expression in vascular smooth muscle cells, while colocalization with macrophages was less pronounced. Conclusions Our study shows that nNOS-α and -γ splice variants are expressed in atherosclerotic plaques of apoE ko mice. nNOS variants colocalized with markers for vascular smooth muscle cells and macrophages but not for endothelial cells. Since nNOS-α is atheroprotective, other nNOS splice variants which differ in enzyme kinetic and subcellular localization may also influence plaque formation.

Mid-infrared reflectivity of experimental atheromas

We report that the pathologic components present within the atheromatous plaques of ApoE knock-out mice can reflect significant amounts of mid-infrared (mid-IR) light. Furthermore, the reflected light spectra contained the unique signatures of a variety of biologic features including those found in unstable or "vulnerable" plaque. This discovery may represent a unique opportunity to develop a new intravascular diagnostic modality that can detect and characterize sites of atherosclerosis.

Abstract 825: Impaired ABCA-1 Mediated Cholesterol Efflux In Hypoxic Macrophages

Atherosclerotic plaques contain large numbers of metabolically active cells that can be deprived of normal oxygen (0 2 ) tension, with levels documented below 10mm Hg (~1% O 2 ) in rabbit and human plaques (vs. normal tissue; 20 –70mmHg, 2.5%–9% O 2 ). Hypoxia has been assumed, but not documented in mouse plaques. Hypoxia stabilizes transcription factor HIF-1α, the master regulator of the cellular response to hypoxia and also involved in reduction-oxidation and inflammatory pathways. Atherosclerotic plaques of apoE knockout mice were immunostained and areas + for HIF-1α as well as two downstream targets (VEGF and Glut-1) were present in increased levels in deeper plaque regions. We next investigated the effect of hypoxia on macrophage function in vitro by incubating murine J774 macrophages in 1% 0 2 in DMEM containing 15% FBS for 24 h and found a 30% increase in cellular total cholesterol (TC) associated with hypoxia. In cells loaded by cyclodextrin-cholesterol, a ~400% increase in TC was observed in hypoxic relative to normoxic cells. Assays using apoAI as a cholesterol acceptor showed that in 1% 0 2 there was a ~90% decrease in ABCA-1 mediated efflux relative to normoxia. In spite of this, ABCA-1 mRNA was increased ~2.5 fold in hypoxic vs. normoxic macrophages. ABCA-1 protein levels by Western analysis remained equal in the 2 conditions. However, by immunofluorescence and confocal microscopy, in sharp distinction to the plasma membrane and cytoplasmic distribution seen in normoxic cells, ABCA-1 localized mainly in a juxtanuclear location in hypoxic macrophages, suggesting impaired trafficking to the membrane. Conclusions: HIF-1α is present in murine plaques, suggesting that they have true hypoxic areas; Hypoxia in vitro promotes macrophage cholesterol accumulation concurrent with ABCA-1 dysfunction; and Hypoxia redistributes ABCA1 from the plasma membrane to the juxtanuclear region.

Defining and Defending Murine Models of Plaque Rupture

There is general agreement that atherosclerotic plaque rupture is a major clinical problem and that finding ways to prevent it would be greatly facilitated by an animal model of plaque rupture. The article by Schwartz et al1 seeks to clarify and define the terms used in descriptions of disrupted atherosclerotic plaques, and then to apply these to murine models in order to assess their similarities to and differences from human ruptured lesions. On the face of it this seems a reasonable thing to do, but it has some serious conceptual problems. The first is the issue of whether it is useful at all to provide definitions of disrupted plaques, especially when these are based on histopathology. The second concerns the transferability of such definitions from human to mouse. I would like also to deal with issues relating to reevaluation of existing mouse models of plaque rupture. What do we gain from framing scientific definitions of real-world phenomena? In the context of the ruptured plaque, Schwartz et al1 argue that this would provide an agreed upon and consistent framework that would make it easier for different labs to compare and contrast their data. There is obviously virtue in this as a general principle in science, but we have to consider the possibility that in the developing field of plaque rupture it may be premature. We do not know why plaques rupture, we do not know whether “plaque erosions” are simply the adjacent manifestations of ruptures that are out of the plane of the section, and we do not know whether “plaque fissures” arise through a different set of processes to plaque ruptures. In other words, this means that we do not know whether ruptures, erosions and fissures are ontogenically similar or different. We are therefore moving beyond the boundaries …

Effects of Rapamycin on the Arterial Inflammatory Response in Atherosclerotic Plaques in Apo-E Knockout Mice

Because of its antiproliferative properties and its known effects on plasma lipids, we evaluated the mechanisms underlying the effect of rapamycin (RPM) on endothelial nitric oxide synthase (eNOS) and matrix metalloproteinases in Apo-E knockout mice. Apo-E −/− mice fed a high-cholesterol diet were given RPM (3 mg/kg per day intraperitoneally) or no treatment for 10 weeks (n = 8 each). Blood was drawn for serum lipid analysis. Protein was extracted from the abdominal aortas for Western immunoblotting and zymography. Cellular localization was assessed by histology and immunohistochemistry. The data, expressed as mean ± SEM, were compared by Student’s t test or analysis of variance (ANOVA). Lipid levels at 10 weeks were similar in both groups except for higher triglyceride levels in RPM-treated animals. RPM-treated mice expressed greater amounts of eNOS and p-eNOS compared with controls ( P < .05). Akt, p-Akt, Caveolin-1, and p-Caveolin-1 were not significantly affected by RPM treatment. RPM treatment was associated with increased activation of pro-MMP-9, a significant decrease in MMP-2 tissue levels, and corresponding increases in TIMP-2 and TIMP-3 expression. The increased expression and phosphorylation of eNOS with RPM appears to be regulated by mechanisms other than Akt or Caveolin-1. Alterations in eNOS expression, in addition to changes in MMP/TIMP ratios and MMP-2 and MMP-9 activation, may partially explain the changes observed in the aorta of treated Apo-E −/− mice induced by RPM.

Chronic Exposure to the Carcinogenic Compound Benzo[a]Pyrene Induces Larger and Phenotypically Different Atherosclerotic Plaques in ApoE-Knockout Mice

Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon with atherogenic and carcinogenic properties. The role of B[a]P in carcinogenesis is well established, and thought to exert via enzymatic activation into reactive metabolites that are capable of binding to the DNA leading to uncontrolled proliferation. However, the mechanism underlying the atherogenic properties of B[a]P is still unclear. Therefore, the effects of chronic B[a]P exposure on atherosclerotic plaque development in apolipoprotein E knockout (apoE-KO) mice were studied. ApoE-KO mice were orally treated with 5 mg/kg/bw B[a]P once per week for 12 or 24 consecutive weeks. Levels of reactive B[a]P metabolites in the arterial tree (from the aortic arch until the iliac artery bifurcations) were high as shown by the level of B[a]P DNA-binding products measured in DNA isolated from the entire aorta (38.9 +/- 4.8 adducts/10(8) nucleotides). Analysis of atherosclerotic lesions in the aortic arch showed no influence of B[a]P on location or number of lesions. Moreover, no increased levels of p53 nuclear protein accumulation or cell proliferation, as detected by immunohistochemistry, were seen in the plaques of the B[a]P-exposed animals. However, the effects of B[a]P on advanced lesions were obvious: advanced plaques were larger and more prone to lipid core development and plaque layering at both 12 and 24 weeks (P < 0.05). In the B[a]P-exposed animals advanced plaques contained more T-lymphocytes and macrophages than in the control animals at both end points (P < 0.05). These data suggest that B[a]P does not initiate atherosclerosis in apoE-KO mice, but accelerates the progression of atherosclerotic plaques via a local inflammatory response.

LRP and senile plaques in Alzheimer’s disease: colocalization with apolipoprotein E and with activated astrocytes

The low density lipoprotein receptor-related protein (LRP) is a multifunctional receptor which is present on senile plaques in Alzheimer’s disease (AD). It is suggested to play an important role in the balance between amyloid beta (Aβ) synthesis and clearance mechanisms. One of its ligands, apolipoprotein E (apoE), is also present on senile plaques and has been implicated as a risk factor for AD, potentially affecting the deposition, fibrillogenesis and clearance of Aβ. Using immunohistochemistry we show that LRP was present only on cored, apoE-containing senile plaques, in both PDAPP transgenic mice and human AD brains. We detected strong LRP staining in neurons and in reactive astrocytes, and immunostaining of membrane-bound LRP showed colocalization with fine astrocytic processes surrounding senile plaques. LRP was not present in plaques in young transgenic mice or in plaques of APOE-knockout mice. As LRP ligands associated with Aβ deposits in AD brain may play an important role in inducing levels of LRP in both neurons and astrocytes, our findings support the idea that apoE might be involved in upregulation of LRP (present in fine astrocytic processes) and act as a local scaffolding protein for LRP and Aβ. The upregulation of LRP would allow increased clearance of LRP ligands as well as clearance of Aβ/ApoE complexes.