eNOS protects from atherosclerosis despite relevant superoxide production by the enzyme in apoE mice

Background

All three nitric oxide synthase (NOS) isoforms are expressed in atherosclerotic plaques. NOS enzymes in general catalyse NO production. However, under conditions of substrate and cofactor deficiency, the enzyme directly catalyse superoxide formation. Considering this alternative chemistry, the effects of NOS on key events in spontaneous hyperlipidemia driven atherosclerosis have not been investigated yet. Here, we evaluate how endothelial nitric oxide synthase (eNOS) modulates leukocyte/endothelial- (L/E) and platelet/endothelial- (P/E) interactions in atherosclerosis and the production of nitric oxide (NO) and superoxide by the enzyme.

Principal Findings

Intravital microscopy (IVM) of carotid arteries revealed significantly increased L/E-interactions in apolipoproteinE/eNOS double knockout mice (apoE^−/−/eNOS^−/−), while P/E-interactions did not differ, compared to apoE^−/−. eNOS deficiency increased macrophage infiltration in carotid arteries and vascular cell adhesion molecule-1 (VCAM-1) expression, both in endothelial and smooth muscle cells. Despite the expression of other NOS isoforms (inducible NOS, iNOS and neuronal NOS, nNOS) in plaques, Electron Spin Resonance (ESR) measurements of NO showed significant contribution of eNOS to total circulating and vascular wall NO production. Pharmacological inhibition and genetic deletion of eNOS reduced vascular superoxide production, indicating uncoupling of the enzyme in apoE^−/− vessels.

Conclusion

Overt plaque formation, increased vascular inflammation and L/E- interactions are associated with significant reduction of superoxide production in apoE^−/−/eNOS^−/− vessels. Therefore, lack of eNOS does not cause an automatic increase in oxidative stress. Uncoupling of eNOS occurs in apoE^−/− atherosclerosis but does not negate the enzyme''s strong protective effects.     

A Murine Model of Obesity With Accelerated Atherosclerosis

The epidemic of obesity sweeping developed nations is accompanied by an increase in atherosclerotic cardiovascular diseases. Dyslipidemia, diabetes, hypertension, and obesity are risk factors for cardiovascular disease. However, delineating the mechanism of obesity‐accelerated atherosclerosis has been hampered by a paucity of animal models. Similar to humans, apolipoprotein E–deficient (apoE^?/?) mice spontaneously develop atherosclerosis over their lifetime. To determine whether apoE^?/? mice would develop obesity with accelerated atherosclerosis, we fed mice diets containing 10 (low fat (LF)) or 60 (high fat (HF)) kcal % from fat for 17 weeks. Mice fed the HF diet had a marked increase in body weight and atherosclerotic lesion formation compared to mice fed the LF diet. There were no significant differences between groups in serum total cholesterol, triglycerides, or leptin concentrations. Plasma concentrations of the acute‐phase reactant serum amyloid A (SAA) are elevated in both obesity and cardiovascular disease. Accordingly, plasma SAA concentrations were increased fourfold (P < 0.01) in mice fed the HF diet. SAA was associated with both pro‐ and antiatherogenic lipoproteins in mice fed the HF diet compared to those fed the LF diet, in which SAA was primarily associated with the antiatherogenic lipoprotein high‐density lipoprotein (HDL). Moreover, SAA was localized with apoB‐containing lipoproteins and biglycan in the vascular wall. Taken together, these data suggest male apoE‐deficient mice are a model of metabolic syndrome and that chronic low level inflammation associated with increased SAA concentrations may mediate atherosclerotic lesion formation.     

The influence of angiotensin-(1–7) Mas receptor agonist (AVE 0991) on mitochondrial proteome in kidneys of apoE knockout mice

Excessive action of angiotensin II on mitochondria has been shown to play an important role in mitochondrial dysfunction, a common feature of atherogenesis and kidney injury. Angiotensin-(1–7)/Mas receptor axis constitutes a countermeasure to the detrimental effects of angiotensin II on AT1 receptors. The aim of the study was to assess the effects of angiotensin-(1–7) peptidomimetic AVE0991 on the kidney mitochondrial proteome in widely used animal model of atherosclerosis (apoE^?/? mice). Proteins changed in apoE^?/? mice belonged to the groups of antioxidant enzymes, apoptosis regulators, inflammatory factors and metabolic enzymes. Importantly, AVE0991 partially reversed atherosclerosis-related changes in apoE^?/? mice.     

The role of hepatic Surf4 in lipoprotein metabolism and the development of atherosclerosis in apoE−/− mice

Elevated plasma levels of low-density lipoprotein-C (LDL-C) increase the risk of atherosclerotic cardiovascular disease. Circulating LDL is derived from very low-density lipoprotein (VLDL) metabolism and cleared by LDL receptor (LDLR). We have previously demonstrated that cargo receptor Surfeit 4 (Surf4) mediates VLDL secretion. Inhibition of hepatic Surf4 impairs VLDL secretion, significantly reduces plasma LDL-C levels, and markedly mitigates the development of atherosclerosis in LDLR knockout (Ldlr^?/?) mice. Here, we investigated the role of Surf4 in lipoprotein metabolism and the development of atherosclerosis in another commonly used mouse model of atherosclerosis, apolipoprotein E knockout (apoE^?/?) mice. Adeno-associated viral shRNA was used to silence Surf4 expression mainly in the liver of apoE^?/? mice. In apoE^?/? mice fed a regular chow diet, knockdown of Surf4 expression significantly reduced triglyceride secretion and plasma levels of non-HDL cholesterol and triglycerides without causing hepatic lipid accumulation or liver damage. When Surf4 was knocked down in apoE^?/? mice fed the Western-type diet, we observed a significant reduction in plasma levels of non-HDL cholesterol, but not triglycerides. Knockdown of Surf4 did not increase hepatic cholesterol and triglyceride levels or cause liver damage, but significantly diminished atherosclerosis lesions. Therefore, our findings indicate the potential of hepatic Surf4 inhibition as a novel therapeutic strategy to reduce the risk of atherosclerotic cardiovascular disease.     

Critical role of caspase-1 in vascular inflammation and development of atherosclerosis in Western diet-fed apolipoprotein E-deficient mice

ObjectiveRecent investigations have suggested that the inflammasome plays a role in the development of vascular inflammation and atherosclerosis; however, its precise role remains controversial. We produced double-deficient mice for apolipoprotien E (Apoe) and caspase-1 (Casp1), a key component molecule of the inflammasome, and investigated the effect of caspase-1 deficiency on vascular inflammation and atherosclerosis.Methods and resultsAtherosclerotic plaque areas in whole aortas and aortic root of Western diet (WD)-fed Apoe^?/?Casp1^?/? mice were significantly reduced compared to those in Apoe^?/? mice. The amount of macrophages and vascular smooth muscle cells in the plaques was also reduced in Apoe^?/?Casp1^?/? mice. No significant differences in plasma lipid profiles and body weight change were observed between these mice. Expression of interleukin (IL)-1β in the plaques as well as plasma levels of IL-1β, IL-1α, IL-6, CCL2, and TNF-α, in Apoe^?/?Casp1^?/? mice were lower than those in Apoe^?/? mice. In vitro experiments showed that calcium phosphate crystals induced caspase-1 activation and secretion of IL-1β and IL-1α in macrophages.ConclusionOur findings suggest that caspase-1 plays a critical role in vascular inflammation and atherosclerosis, and that modulation of caspase-1 could be a potential target for prevention and treatment of atherosclerosis.     

Mouse mesenchymal stem cells from bone marrow differentiate into smooth muscle cells by induction of plaque-derived smooth muscle cells

AimThe present study aimed to elucidate the mechanism by which bone marrow mesenchymal stem cells (BMSCs) differentiate into smooth muscle cells (SMCs) in atherosclerosis.Main methodsWe isolated mouse BMSCs and incubated them in conditioned medium from plaque-derived SMCs (SMC-CM) and analyzed growth factors from media. BMSCs were treated with different media and harvested at continuous time points for investigating the ability to differentiate toward SMCs. Next, BMSCs of green fluorescence protein (GFP) mice were transplanted into apolipoprotein E^?/? (apoE^?/?) mice fed on western type diet for 12 weeks. In vivo efficacy of BMSCs was investigated.Key findingsAfter being cultured using SMC-CM, hepatocyte growth factor (HGF) was abundantly secreted into the medium by BMSCs with time. BMSCs had increased expression of HGF receptor c-met and SMC-specific markers while they also displayed SMC characteristic ‘hill and valley-like’ appearance with an SMC ultra-structure including actin filaments and dense bodies. In vivo-grafted BMSCs aggravated atherosclerotic lesions and inflammation but ameliorated fibrosis in aorta while they displayed higher expression levels of c-met and early SMC-specific markers but not late-stage markers in aorta. They also demonstrated greater secretion of HGF in the aorta of apoE^?/? mice. Furthermore, when BMSCs were treated with HGF blocking antibody, they lost the ability to differentiate to SMCs.SignificanceHGF from local SMCs plays an important role for the differentiation of homing BMSCs.     

A Deficiency of Herp,an Endoplasmic Reticulum Stress Protein,Suppresses Atherosclerosis in ApoE Knockout Mice by Attenuating Inflammatory Responses

Herp was originally identified as an endoplasmic reticulum (ER) stress protein in vascular endothelial cells. ER stress is induced in atherosclerotic lesions, but it is not known whether Herp plays any role in the development of atherosclerosis. To address this question, we generated Herp- and apolipoprotein E (apoE)-deficient mice (Herp^−/−; apoE^−/− mice) by crossbreeding Herp^−/− mice and apoE^−/− mice. Herp was expressed in the endothelial cells and medial smooth muscle cells of the aorta, as well as in a subset of macrophages in the atherosclerotic lesions in apoE^−/− mice, while there was no expression of Herp in the Herp^−/−; apoE^−/− mice. The doubly deficient mice developed significantly fewer atherosclerotic lesions than the apoE^−/− mice at 36 and 72 weeks of age, whereas the plasma levels of cholesterol and triglycerides were not significantly different between the strains. The plasma levels of non-esterified fatty acids were significantly lower in the Herp^−/−; apoE^−/− mice when they were eight and 16 weeks old. The gene expression levels of ER stress response proteins (GRP78 and CHOP) and inflammatory cytokines (IL-1β, IL-6, TNF-α and MCP-1) in the aorta were significantly lower in Herp^−/−; apoE^−/− mice than in apoE^−/− mice, suggesting that Herp mediated ER stress-induced inflammation. In fact, peritoneal macrophages isolated from Herp-deficient mice and RAW264.7 macrophages in which Herp was eliminated with a siRNA expressed lower levels of mRNA for inflammatory cytokines when they were treated with tunicamycin. Herp deficiency affected the major mediators of the unfolded protein response, including IRE1 and PERK, but not ATF6. These findings suggest that a deficiency of Herp suppressed the development of atherosclerosis by attenuating the ER stress-induced inflammatory reactions.     

Fargesin alleviates atherosclerosis by promoting reverse cholesterol transport and reducing inflammatory response

Background and aimsFargesin mainly functions in the improvement of lipid metabolism and the inhibition of inflammation, but the role of fargesin in atherogenesis and the molecular mechanisms have not been defined. We aimed to explore if and how fargesin affects atherosclerosis by regulating lipid metabolism and inflammatory response.Methods and resultsApoE^−/− mice were fed a high-fat diet to form atherosclerotic plaques and then administrated with fargesin or saline via gavage. Oil Red O, HE and Masson staining were performed to assess atherosclerostic plaques in apoE^−/− mice. [³H] labeled cholesterol was used to detect cholesterol efflux and reverse cholesterol transport (RCT) efficiency. Enzymatic methods were performed to analyze plasma lipid profile in apoE^−/− mice. Immunohistochemistry was used to analyze macrophage infiltration. THP-1-derived macrophages were incubated with fargesin or not. Both Western blot and qRT-PCR were applied to detect target gene expression. Oil Red O staining was applied to examine lipid accumulation in THP-1-derived macrophages. ELISA and qRT-PCR were used to examine the levels of inflammatory mediotors. We found that fargesin reduced atherosclerotic lesions by elevating efficiency of RCT and decreasing inflammatory response via upregulation of ABCA1 and ABCG1 expression in apoE^−/− mice. Further, fargesin reduced lipid accumulation in THP-1-derived macrophages. Besides, fargesin increased phosphorylation of CEBPα in Ser21 and then upregulated LXRα, ABCA1 and ABCG1 expression in THP-1-derived macrophages. In addition, fargesin could reduce ox-LDL-induced inflammatory response by inactivation of the TLR4/NF-κB pathway.ConclusionThese results suggest that fargesin inhibits atherosclerosis by promoting RCT process and reducing inflammatory response via CEBPα^S21/LXRα and TLR4/NF-κB pathways, respectively.     

Impaired Vitamin D Signaling in Endothelial Cell Leads to an Enhanced Leukocyte-Endothelium Interplay: Implications for Atherosclerosis Development

Endothelial cell activation leading to leukocyte recruitment and adhesion plays an essential role in the initiation and progression of atherosclerosis. Vitamin D has cardioprotective actions, while its deficiency is a risk factor for the progression of cardiovascular damage. Our aim was to assess the role of basal levels of vitamin D receptor (VDR) on the early leukocyte recruitment and related endothelial cell-adhesion-molecule expression, as essential prerequisites for the onset of atherosclerosis. Knockdown of VDR in endothelial cells (shVDR) led to endothelial cell activation, characterized by upregulation of VCAM-1, ICAM-1 and IL-6, decreased peripheral blood mononuclear cell (PBMC) rolling velocity and increased PBMC rolling flux and adhesion to the endothelium. shVDR cells showed decreased IκBα levels and accumulation of p65 in the nucleus compared to shRNA controls. Inhibition of NF-κB activation with super-repressor IκBα blunted all signs of endothelial cell activation caused by downregulation of VDR in endothelial cells. In vivo, deletion of VDR led to significantly larger aortic arch and aortic root lesions in apoE^-/- mice, with higher macrophage content. apoE^-/-VDR^-/-mice showed higher aortic expression of VCAM-1, ICAM-1 and IL-6 when compared to apoE^-/-VDR^+/+ mice. Our data demonstrate that lack of VDR signaling in endothelial cells leads to a state of endothelial activation with increased leukocyte-endothelial cell interactions that may contribute to the more severe plaque accumulation observed in apoE^-/-VDR^-/- mice. The results reveal an important role for basal levels of endothelial VDR in limiting endothelial cell inflammation and atherosclerosis.     

AnxA5 reduces plaque inflammation of advanced atherosclerotic lesions in apoE−/− mice

Annexin A5 (AnxA5) exerts anti‐inflammatory, anticoagulant and anti‐apoptotic effects through binding cell surface expressed phosphatidylserine. The actions of AnxA5 on atherosclerosis are incompletely understood. We investigated effects of exogenous AnxA5 on plaque morphology and phenotype of advanced atherosclerotic lesions in apoE^?/? mice. Advanced atherosclerotic lesions were induced in 12 weeks old Western type diet fed apoE^?/? mice using a collar placement around the carotid artery. After 5 weeks mice were injected either with AnxA5 (n = 8) or vehicle for another 4 weeks. AnxA5 reduced plaque macrophage content both in the intima (59% reduction, P < 0.05) and media (73% reduction, P < 0.01) of advanced atherosclerotic lesions of the carotid artery. These findings corroborated with advanced lesions of the aortic arch, where a 67% reduction in plaque macrophage content was observed with AnxA5 compared to controls (P < 0.01). AnxA5 did not change lesion extension, plaque apoptosis, collagen content, smooth muscle cell content or acellular plaque composition after 4 weeks of treatment as determined by immunohistochemistry in advanced carotid lesions. In vitro, AnxA5 exhibited anti‐inflammatory effects in macrophages and a flow chamber based assay demonstrated that AnxA5 significantly inhibited capture, rolling, adhesion as well as transmigration of peripheral blood mononuclear cells on a TNF‐α‐activated endothelial cell layer. In conclusion, short‐term treatment with AnxA5 reduces plaque inflammation of advanced lesions in apoE^?/? mice likely through interfering with recruitment and activation of monocytes to the inflamed lesion site. Suppressing chronic inflammation by targeting exposed phosphatidylserine may become a viable strategy to treat patients suffering from advanced atherosclerosis.     

Atheroprotective Effect of Oleoylethanolamide (OEA) Targeting Oxidized LDL

Dietary fat-derived lipid oleoylethanolamide (OEA) has shown to modulate lipid metabolism through a peroxisome proliferator-activated receptor-alpha (PPAR-α)-mediated mechanism. In our study, we further demonstrated that OEA, as an atheroprotective agent, modulated the atherosclerotic plaques development. In vitro studies showed that OEA antagonized oxidized LDL (ox-LDL)-induced vascular endothelial cell proliferation and vascular smooth muscle cell migration, and suppressed lipopolysaccharide (LPS)-induced LDL modification and inflammation. In vivo studies, atherosclerosis animals were established using balloon-aortic denudation (BAD) rats and ApoE^-/- mice fed with high-caloric diet (HCD) for 17 or 14 weeks respectively, and atherosclerotic plaques were evaluated by oil red staining. The administration of OEA (5 mg/kg/day, intraperitoneal injection, i.p.) prevented or attenuated the formation of atherosclerotic plaques in HCD-BAD rats or HCD-ApoE^−/− mice. Gene expression analysis of vessel tissues from these animals showed that OEA induced the mRNA expressions of PPAR-α and downregulated the expression of M-CFS, an atherosclerotic marker, and genes involved in oxidation and inflammation, including iNOS, COX-2, TNF-α and IL-6. Collectively, our results suggested that OEA exerted a pharmacological effect on modulating atherosclerotic plaque formation through the inhibition of LDL modification in vascular system and therefore be a potential candidate for anti-atherosclerosis drug.     

NOX1 deficiency in apolipoprotein E-knockout mice is associated with elevated plasma lipids and enhanced atherosclerosis

Abstract

Nicotinamide adenine dinucleotide phosphate oxidases (NOX) are enzymes that generate reactive oxygen species (ROS). NOX2 activity in the vascular wall is elevated in hypercholesterolemia, and contributes to oxidative stress and atherogenesis. Here we examined the role of another NOX isoform, NOX1, in atherogenesis in apolipoprotein E-knockout (APOE^?/?) mice fed a Western diet for 14 weeks. Although NOX1 mRNA expression was unchanged in aortas from APOE^?/? versus wild-type mice, expression of the NOX1-specific organizer, NOXO1, was diminished, consistent with an overall reduction in NOX1 activity in APOE^?/? mice. To examine the impact of a further reduction in NOX1 activity, APOE^?/? mice were crossed with NOX1^?/y mice to generate NOX1^?/y/APOE^?/? double-knockouts. NOX1 deficiency in APOE^?/? mice was associated with 30–50% higher plasma very-low-density lipoprotein (VLDL)/LDL and triglyceride levels (P < 0.01). Vascular ROS levels were also elevated by twofold in NOX1^?/y/APOE^?/? versus APOE^?/? mice (P < 0.05), despite no changes in expression of other NOX subunits. Although en face analysis of the descending aorta revealed no differences in plaque area between NOX1^?/y/APOE^?/? and APOE^?/? mice, intimal thickening in the aortic sinus was increased by 40% (P < 0.05) in the double-knockouts. Moreover, NOX1 deficiency was associated with a less stable plaque phenotype; aortic sinus lesions contained 60% less collagen (P < 0.01), 40% less smooth muscle (P < 0.01), and 2.5-fold higher levels of matrix metalloproteinase-9 (P < 0.001) than lesions in APOE^?/? mice. Thus, these data, which suggest a protective role for NOX1 against hyperlipidemia and atherosclerosis in APOE^?/? mice, highlight the complex and contrasting roles of different NOX isoforms (e.g., NOX2 versus NOX1) in vascular pathology.     

Comparison between genetic and pharmaceutical disruption of Ldlr expression for the development of atherosclerosis

Antisense oligonucleotides (ASOs) against Ldl receptor (Ldlr-ASO) represent a promising strategy to promote hypercholesterolemic atherosclerosis in animal models without the need for complex breeding strategies. Here, we sought to characterize and contrast atherosclerosis in mice given Ldlr-ASO with those bearing genetic Ldlr deficiency. To promote atherosclerosis, male and female C57Bl6/J mice were either given weekly injections of Ldlr-ASO (5 mg/kg once per week) or genetically deficient in Ldlr (Ldlr^?/?). Mice consumed either standard rodent chow or a diet high in saturated fat and sucrose with 0.15% added cholesterol for 16 weeks. While both models of Ldlr deficiency promoted hypercholesterolemia, Ldlr^?/? mice exhibited nearly 2-fold higher cholesterol levels than Ldlr-ASO mice, reflected by increased VLDL and LDL levels. Consistent with this, the en face atherosclerotic lesion area was 3-fold and 3.6-fold greater in male and female mice with genetic Ldlr deficiency, respectively, as compared with the modest atherosclerosis observed following Ldlr-ASO treatment. Aortic sinus lesion sizes, fibrosis, smooth muscle actin, and necrotic core areas were also larger in Ldlr^?/? mice, suggesting a more advanced phenotype. Despite a more modest effect on hypercholesterolemia, Ldlr-ASO induced greater hepatic inflammatory gene expression, macrophage accumulation, and histological lobular inflammation than was observed in Ldlr^?/? mice. We conclude Ldlr-ASO is a promising tool for the generation of complex rodent models with which to study atherosclerosis but does not promote comparable levels of hypercholesterolemia or atherosclerosis as Ldlr^?/? mice and increases hepatic inflammation. Thus, genetic Ldlr deficiency may be a superior model, depending on the proposed use.     

Angiopoietin-1 aggravates atherosclerosis by inhibiting cholesterol efflux and promoting inflammatory response

ObjectiveAngiopoietin-1 (Ang-1), a secreted protein, mainly regulates angiogenesis. Ang-1 has been shown to promote the development of atherosclerosis, whereas little is known about its effects on lipid metabolism and inflammation in this process.MethodAng-1 was transfected into ApoE^−/− mice via lentiviral vector or incubated with THP-1 derived macrophages. Oil red O and HE staining were performed to measure the size of atherosclerotic plaques in ApoE^−/− mice. Immunofluorescence was employed to show the expression of target proteins in aorta. [³H] labeled cholesterol was performed to examine the efficiency of cholesterol efflux and reverse cholesterol transport (RCT) both in vivo and vitro. Western blot and qPCR were used to quantify target proteins both in vivo and vitro. ELISA detected the levels of pro-inflammatory cytokines in mouse peritoneal macrophage.ResultsOur data showed that Ang-1 augmented atherosclerotic plaques formation and inhibited cholesterol efflux. The binding of Ang-1 to Tie2 resulted in downregulation of LXRα, ABCA1 and ABCG1 expression via inhibiting the translocation of TFE3 into nucleus. In addition, Ang-1 decreased serum HDL-C levels and reduced reverse cholesterol transport (RCT) in ApoE−/− mice. Furthermore, Ang-1 induced lipid accumulation followed by increasing TNF-α, IL-6, IL-1β,and MCP-1 produced by MPMs, as well as inducing M1 phenotype macrophage marker iNOS and CD86 expression in aorta of ApoE^−/− mice.ConclusionAng-1 has an adverse effect on cholesterol efflux by decreasing the expression of ABCA1 and ABCG1 via Tie2/TFE3/LXRα pathway, thereby promoting inflammation and accelerating atherosclerosis progression.     

Targeting annexin A7 by a small molecule suppressed the activity of phosphatidylcholine-specific phospholipase C in vascular endothelial cells and inhibited atherosclerosis in apolipoprotein E?/? mice

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a key factor in apoptosis and autophagy of vascular endothelial cells (VECs), and involved in atherosclerosis in apolipoprotein E^−/− (apoE^−/−) mice. But the endogenous regulators of PC-PLC are not known. We recently found a small chemical molecule (6-amino-2, 3-dihydro-3-hydroxymethyl-1, 4-benzoxazine, ABO) that could inhibit oxidized low-density lipoprotein (oxLDL)-induced apoptosis and promote autophagy in VECs, and further identified ABO as an inhibitor of annexin A7 (ANXA7) GTPase. Based on these findings, we hypothesize that ANXA7 is an endogenous regulator of PC-PLC, and targeting ANXA7 by ABO may inhibit atherosclerosis in apoE^−/− mice. In this study, we tested our hypothesis. The results showed that ABO suppressed oxLDL-induced increase of PC-PLC level and activity and promoted the co-localization of ANXA7 and PC-PLC in VECs. The experiments of ANXA7 knockdown and overexpression demonstrated that the action of ABO was ANXA7-dependent in cultured VECs. To investigate the relation of ANXA7 with PC-PLC in atherosclerosis, apoE^−/− mice fed with a western diet were treated with 50 or 100 mg/kg/day ABO. The results showed that ABO decreased PC-PLC levels in the mouse aortic endothelium and PC-PLC activity in serum, and enhanced the protein levels of ANXA7 in the mouse aortic endothelium. Furthermore, both dosages of ABO significantly enhanced autophagy and reduced apoptosis in the mouse aortic endothelium. As a result, ABO significantly reduced atherosclerotic plaque area and effectively preserved a stable plaques phenotype, including reduced lipid deposition and pro-inflammatory macrophages, increased anti-inflammatory macrophages, collagen content and smooth muscle cells, and less cell death in the plaques. In conclusion, ANXA7 was an endogenous regulator of PC-PLC, and targeting ANXA7 by ABO inhibited atherosclerosis in apoE^−/− mice.     

Effects of Exogenous Recombinant APC in Mouse Models of Ischemia Reperfusion Injury and of Atherosclerosis

Activated protein C (APC) is a serine protease that has both anticoagulant and cytoprotective properties. The cytoprotective effects are protease activated receptor 1 (PAR-1) and endothelial protein C receptor (EPCR) dependent and likely underlie protective effects of APC in animal models of sepsis, myocardial infarction and ischemic stroke. S360A-(A)PC, a variant (A)PC that has no catalytic activity, binds EPCR and shifts pro-inflammatory signaling of the thrombin-PAR-1 complex to anti-inflammatory signaling. In this study we investigated effects of human (h)wt-PC, hS360A-PC, hwt-APC and hS360A-APC in acute (mouse model of acute myocardial ischemia/reperfusion (I/R) injury) and chronic inflammation (apoE^−/− mouse model of atherosclerosis). All h(A)PC variants significantly reduced myocardial infarct area (p<0.05) following I/R injury. IL-6 levels in heart homogenates did not differ significantly between sham, placebo and treatment groups in I/R injury. None of the h(A)PC variants decreased number and size of atherosclerotic plaques in apoE^−/− mice. Only hS360A-APC slightly affected phenotype of plaques. IL-6 levels in plasma were significantly (p<0.001) decreased in hwt-APC and hS360A-PC treated mice. In the last group levels of monocyte chemotactic protein 1 (MCP-1) were significantly increased (p<0.05). In this study we show that both hwt and hS360A-(A)PC protect against acute myocardial I/R injury, which implies that protection from I/R injury is independent of the proteolytic activity of APC. However, in the chronic atherosclerosis model hwt and hS360-(A)PC had only minor effects. When the dose, species and mode of (A)PC administration will be adjusted, we believe that (A)PC will have potential to influence development of chronic inflammation as occurring during atherosclerosis as well.     

TLR4/NF-κB Signaling Contributes to Chronic Unpredictable Mild Stress-Induced Atherosclerosis in ApoE-/- Mice

Objective

Chronic stress is an important risk factor for atherosclerotic diseases. Our previous studies have shown that chronic unpredictable mild stress (CUMS) accelerates atherosclerosis and up-regulates TLR4/NF-κB expression in apoE^-/- mice. However, TLR4/NF-κB signaling whether directly contributes to the development of atherosclerosis in CUMS mice is unclear. We hypothesized that the interference of TLR4/NF-κB can ameliorate CUMS-induced inflammation and atherosclerosis in apoE^-/- mice.

Methods

ApoE^-/- mice were exposed to 12 weeks CUMS. Ad-siRNA TLR4 was given by tail vein injection (10 μl/mouse, every 5 days), and PDTC (an inhibitor of NF-κB) was given by intraperitoneal injection (60 mg/kg, once a day). Plasma corticosterone concentrations were determined by solid-phase ¹²⁵I radioimmunoassay. Atherosclerosis lesions in aortic sinuses were evaluated and quantified by IMAGEPRO PLUS. Western blotting was used to detect the expression of TLR4, NF-κB, and IL-1β in aortas of the mice. Plasma lipid profiles, IL-1β, TNF-α, and MCP-1 were measured by ELISA.

Results

Our results indicated that CUMS apoE^-/- mice treatment with siRNA TLR4 significantly decreased atherosclerosis and down-regulated TLR4, NF-κB, and inflammatory cytokines. PDTC also remarkably reduced atherosclerosis and the levels of IL-1β, TNF-α and MCP-1 in plasma. However, Treatment with siRNA TLR4 or PDTC had no effect on plasma corticosterone levels, and lipid profiles.

Conclusions

TLR4/NF-κB pathway may participate in CUMS-induced atherosclerosis through activation of proinflammatory cytokines in apoE^-/- mice. Our data may provide a new potential therapeutic target for prevention of CUMS -induced atherosclerosis.     

Macrophage-derived apoESendai suppresses atherosclerosis while causing lipoprotein glomerulopathy in hyperlipidemic mice

Lipoprotein glomerulopathy (LPG) is a renal disease often accompanied by dyslipidemia and increased serum apoE levels. apoE_Sendai (Arg145Pro), a rare mutant based on the apoE3 sequence carrying an apoE2 charge, causes LPG in humans and transgenic mice, but its effects on the artery wall are unknown. Macrophage expression of apoE_Sendai may also directly influence renal and arterial homeostasis. We investigated the effects of macrophage-expressed apoE_Sendai in apoE^−/− mice with or without LDL receptor (LDLR). Murine bone marrow transduced to express apoE2, apoE3, or apoE_Sendai was transplanted into lethally irradiated mice. Macrophage apoE_Sendai expression reduced aortic lesion size and inflammation by 32 and 28%, respectively, compared with apoE2 in apoE^−/− recipients. No differences in lesion size or inflammation were found between apoE_Sendai and apoE3 in apoE^−/− recipients. Macrophage apoE_Sendai expression also reduced aortic lesion size by 18% and inflammation by 29% compared with apoE2 in apoE^−/−/LDLR^−/− recipients. Glomerular lesions compatible with LPG with increased mesangial matrix, extracellular lipid accumulation, and focal mesangiolysis were only observed in apoE^−/−/LDLR^−/− mice expressing apoE_Sendai. Thus, macrophage expression of apoE_Sendai protects against atherosclerosis while causing lipoprotein glomerulopathy. This is the first demonstration of an apoprotein variant having opposing effects on vascular and renal homeostasis.     

Extravascular inflammation does not increase atherosclerosis in apoE-deficient mice

There is much speculation whether extravascular inflammation accelerates atherosclerosis. We tested this hypothesis in apoE^−/− mice using three well-characterized models of non-autoimmune chronic inflammation: croton oil-induced skin inflammation, Aspergillus fumigatus antigen-induced allergic lung disease, and A. fumigatus antigen-induced peritonitis. The croton oil model produced recurrent inflammatory skin ulceration, and marked increases in plasma levels of IL-6 and serum amyloid A (SAA). The allergic lung disease model showed strong local inflammation with eosinophilic infiltration and serum IgE induction. The recurrent peritonitis model was accompanied by mild elevation in plasma SAA levels. Aortic atherosclerosis was quantified by computer-assisted morphometry of en face arteries in apoE^−/− mice at 34 weeks for the croton oil model, 26 and 42 weeks for the allergic lung disease model, and 26 weeks for the peritonitis model. We found that all three forms of chronic extravascular inflammation had no effect on the rate of atherosclerosis development.