Autophagy dysfunction and regulatory cystatin C in macrophage death of atherosclerosis

Autophagy dysfunction in mouse atherosclerosis models has been associated with increased lipid accumulation, apoptosis and inflammation. Expression of cystatin C (CysC) is decreased in human atheroma, and CysC deficiency enhances atherosclerosis in mice. Here, we first investigated the association of autophagy and CysC expression levels with atheroma plaque severity in human atherosclerotic lesions. We found that autophagy proteins Atg5 and LC3β in advanced human carotid atherosclerotic lesions are decreased, while markers of dysfunctional autophagy p62/SQSTM1 and ubiquitin are increased together with elevated levels of lipid accumulation and apoptosis. The expressions of LC3β and Atg5 were positively associated with CysC expression. Second, we investigated whether CysC expression is involved in autophagy in atherosclerotic apoE‐deficient mice, demonstrating that CysC deficiency (CysC^?/?) in these mice results in reduction of Atg5 and LC3β levels and induction of apoptosis. Third, macrophages isolated from CysC^?/? mice displayed increased levels of p62/SQSTM1 and higher sensitivity to 7‐oxysterol‐mediated lysosomal membrane destabilization and apoptosis. Finally, CysC treatment minimized oxysterol‐mediated cellular lipid accumulation. We conclude that autophagy dysfunction is a characteristic of advanced human atherosclerotic lesions and is associated with reduced levels of CysC. The deficiency of CysC causes autophagy dysfunction and apoptosis in macrophages and apoE‐deficient mice. The results indicate that CysC plays an important regulatory role in combating cell death via the autophagic pathway in atherosclerosis.     

Complement factor C5a induces atherosclerotic plaque disruptions

Complement factor C5a and its receptor C5aR are expressed in vulnerable atherosclerotic plaques; however, a causal relation between C5a and plaque rupture has not been established yet. Accelerated atherosclerosis was induced by placing vein grafts in male apoE^?/? mice. After 24 days, when advanced plaques had developed, C5a or PBS was applied locally at the lesion site in a pluronic gel. Three days later mice were killed to examine the acute effect of C5a on late stage atherosclerosis. A significant increase in C5aR in the plaque was detectable in mice treated with C5a. Lesion size and plaque morphology did not differ between treatment groups, but interestingly, local treatment with C5a resulted in a striking increase in the amount of plaque disruptions with concomitant intraplaque haemorrhage. To identify the potential underlying mechanisms, smooth muscle cells and endothelial cells were treated in vitro with C5a. Both cell types revealed a marked increase in apoptosis after stimulation with C5a, which may contribute to lesion instability in vivo. Indeed, apoptosis within the plaque was seen to be significantly increased after C5a treatment. We here demonstrate a causal role for C5a in atherosclerotic plaque disruptions, probably by inducing apoptosis. Therefore, intervention in complement factor C5a signalling may be a promising target in the prevention of acute atherosclerotic complications.     

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.     

Deletion of junctional adhesion molecule A from platelets increases early‐stage neointima formation after wire injury in hyperlipidemic mice

Platelets play an important role in the pathogenesis of vascular remodelling after injury. Junctional adhesion molecule A (JAM‐A) was recently described to regulate platelet activation. Specific deletion of JAM‐A from platelets resulted in increased reactivity and in accelerated progression of atherosclerosis. The aim of this study was to investigate the specific contribution of platelet‐derived JAM‐A to neointima formation after vascular injury. Mice with or without platelet‐specific (tr)JAM‐A‐deficiency in an apolipoprotein e (apoe^?/?) background underwent wire‐induced injury of the common carotid artery. Ex vivo imaging by two‐photon microscopy revealed increased platelet coverage at the site of injury in trJAM‐A‐deficient mice. Cell recruitment assays showed increased adhesion of monocytic cells to activated JAM‐A‐deficient platelets than to control platelets. Inhibition of α_Mβ₂ or GPIbα, but not of CD62P, suppressed those differences. Up to 4 weeks after wire injury, intimal neoplasia and neointimal cellular content were analysed. Neointimal lesion area was increased in trJAM‐A^?/? apoe^?/? mice and the lesions showed an increased macrophage accumulation and proliferating smooth muscle cells compared with trJAM‐A^+/+ apoe^?/? littermates 2 weeks, but not 4 weeks after injury. Re‐endothelialization was decreased in trJAM‐A^?/? apoe^?/? mice compared with controls 2 weeks after injury, yet it was complete in both groups after 4 weeks. A platelet gain of function by deletion of JAM‐A accelerates neointima formation only during earlier phases after vascular injury, through an increased recruitment of mononuclear cells. Thus, the contribution of platelets might become less important when neointima formation progresses to later stages.     

Interleukin-17 deficiency reduced vascular inflammation and development of atherosclerosis in Western diet-induced apoE-deficient mice

ObjectiveSeveral reports describe the role of interleukin (IL)-17 in the development of atherosclerosis; however, its precise role remains controversial. We generated double-deficient mice for apolipoprotein E (apoE) and IL-17 (apoE^?/?IL-17^?/? mice) and investigated the effect of IL-17 deficiency on vascular inflammation and atherosclerosis.Methods and resultsAtherosclerotic plaque areas in apoE^?/?IL-17^?/? mice fed a Western diet (WD) were significantly reduced compared with those in apoE^?/? mice. No significant differences in plasma lipid profiles were observed between apoE^?/? and apoE^?/?IL-17^?/? mice. The number of infiltrated macrophages in the plaques was significantly decreased in WD-fed apoE^?/?IL-17^?/? mice compared with WD-fed apoE^?/? mice, whereas vascular smooth muscle cell content was not altered by IL-17 deficiency. Expression of inflammatory cytokines (MCP-1, IL-1β, IL-6, IFN-γ, and IL-12 p40) and scavenger receptors (Msr-1, Scarb1, and Olr1) in the plaques was inhibited in WD-fed apoE^?/?IL-17^?/? mice. Furthermore, expression of inducible nitric oxide (M1 marker) and arginase-1 (M2 marker) was inhibited in WD-fed apoE^?/?IL-17^?/? mice.ConclusionOur results indicate that IL-17 deficiency reduces vascular inflammation and atherosclerosis and that modulation of IL-17 could be a potential target for prevention and treatment of atherosclerosis.     

Activation of CXCR7 promotes endothelial repair and reduces the carotid atherosclerotic lesions through inhibition of pyroptosis signaling pathways

Endothelial injuries, including cell pyroptosis, are ongoing inflammatory processes with key roles in atherosclerosis development. Our previous report showed that the chemokine CXCL12 and its receptor CXCR7 are associated with the proliferation and angiogenesis of endothelial cells. Nevertheless, the mechanism underlying these effects on atherosclerotic lesions, especially on endothelial dysfunction, remains unknown. Here, we demonstrated that CXCR7 was upregulated in human carotid atherosclerotic plaques, apolipoprotein E knockout (ApoE^?/?) mice fed with a high‐fat diet (HFD), and oxidized lipopolysaccharide‐treated (ox‐LDL) human umbilical vein endothelial cells (HUVECs). Further, the activation of CXCR7 reversed ox‐LDL‐induced HUVEC dysfunction, such as migration, tube formation, and cell pyroptosis; all of these protective effects were alleviated by inhibition of CXCR7. The NOD‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasomes were also elevated in human carotid atherosclerotic plaques, ApoE^?/? mice fed with HFD, and ox‐LDL‐injured HUVECs by regulation of caspase‐1 and interleukin (IL)‐1β expression. The activation of CXCR7 by TC14012 led to a decrease in atherosclerotic lesions in ApoE^?/? mice fed with HFD. TC14012 also inhibited the expression of the NLRP3 inflammasome signaling pathway in vivo. In conclusion, our study suggests that CXCR7 plays an important role in regulating NLRP3 inflammasome‐modulated pyroptosis in HUVECs, providing a potential novel therapy for atherosclerosis.     

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.     

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 Salmon Protein Hydrolysate Exerts Lipid-Independent Anti-Atherosclerotic Activity in ApoE-Deficient Mice

Fish consumption is considered health beneficial as it decreases cardiovascular disease (CVD)-risk through effects on plasma lipids and inflammation. We investigated a salmon protein hydrolysate (SPH) that is hypothesized to influence lipid metabolism and to have anti-atherosclerotic and anti-inflammatory properties. 24 female apolipoprotein (apo) E^−/− mice were divided into two groups and fed a high-fat diet with or without 5% (w/w) SPH for 12 weeks. The atherosclerotic plaque area in aortic sinus and arch, plasma lipid profile, fatty acid composition, hepatic enzyme activities and gene expression were determined. A significantly reduced atherosclerotic plaque area in the aortic arch and aortic sinus was found in the 12 apoE^−/− mice fed 5% SPH for 12 weeks compared to the 12 casein-fed control mice. Immunohistochemical characterization of atherosclerotic lesions in aortic sinus displayed no differences in plaque composition between mice fed SPH compared to controls. However, reduced mRNA level of Icam1 in the aortic arch was found. The plasma content of arachidonic acid (C20∶4n-6) and oleic acid (C18∶1n-9) were increased and decreased, respectively. SPH-feeding decreased the plasma concentration of IL-1β, IL-6, TNF-α and GM-CSF, whereas plasma cholesterol and triacylglycerols (TAG) were unchanged, accompanied by unchanged mitochondrial fatty acid oxidation and acyl-CoA:cholesterol acyltransferase (ACAT)-activity. These data show that a 5% (w/w) SPH diet reduces atherosclerosis in apoE^−/− mice and attenuate risk factors related to atherosclerotic disorders by acting both at vascular and systemic levels, and not directly related to changes in plasma lipids or fatty acids.     

The anti-inflammatory effects of exercise training promote atherosclerotic plaque stabilization in apolipoprotein E knockout mice with diabetic atherosclerosis

Physical exercise is the cornerstone of cardiovascular disease treatment. The present study investigated whether exercise training affects atherosclerotic plaque composition through the modification of inflammatoryrelated pathways in apolipoprotein E knockout (apoE^−/−) mice with diabetic atherosclerosis. Forty-five male apoE^−/− mice were randomized into three equivalent (n=15) groups: control (CO), sedentary (SED), and exercise (EX). Diabetes was induced by streptozotocin administration. High-fat diet was administered to all groups for 12 weeks. Afterwards, CO mice were euthanatized, while the sedentary and exercise groups continued high-fat diet for 6 additional weeks. Exercising mice followed an exercise program on motorizedtreadmill (5 times/week, 60 min/session). Then, blood samples and atherosclerotic plaques in the aortic root were examined. A considerable (P<0.001) regression of the atherosclerotic lesions was observed in the exercise group (180.339±75.613×10³µm²) compared to the control (325.485±72.302×10³ µm²) and sedentary (340.188±159.108×10³µm²) groups. We found decreased macrophages, matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-8 and interleukin-6 (IL-6) concentrations (P<0.05) in the atherosclerotic plaques of the exercise group. Compared to both control and sedentary groups, exercise training significantly increased collagen (P<0.05), elastin (P<0.001), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) (P<0.001) content in the atherosclerotic plaques. Those effects paralleled with increased fibrous cap thickness and less internal elastic lamina ruptures after exercise training (P<0.05), while body-weight and lipid parameters did not significantly change. Plasma MMP-2 and MMP-3 concentrations in atherosclerotic tissues followed a similar trend. From our study we can conclude that exercise training reduces and stabilizes atherosclerotic lesions in apoE^−/− mice with diabetic atherosclerosis. A favorable modification of the inflammatory regulators seems to explain those beneficial effects.Key words: diabetes, atherosclerosis, exercise, matrix metalloproteinases, plaque stability.     

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.     

Aldehyde dehydrogenase 2 deficiency promotes atherosclerotic plaque instability through accelerating mitochondrial ROS-mediated vascular smooth muscle cell senescence

Previous evidence has indicated a beneficial role for aldehyde dehydrogenase 2 (ALDH2) in suppressing atherosclerotic plaque progression and instability. However, the underlying mechanism remains somewhat elusive. This study was designed to examine the effect of ALDH2 deficiency on high-cholesterol diet-induced atherosclerotic plaque progression and plaque vulnerability in atherosclerosis-prone ApoE knockout (ApoE^?/?) mice with a focus on foam cell formation in macrophages and senescence of vascular smooth muscle cells (VSMCs). Serum lipid profile, plaque progression, and plaque vulnerability were examined in ApoE^?/? and ALDH2/ApoE double knockout (ALDH2^?/?ApoE^?/?) mice after high-cholesterol diet intake for 8 weeks. ALDH2 deficiency increased the serum levels of triglycerides while it decreased levels of total cholesterol and high-density lipoprotein cholesterol. Unexpectedly, ALDH2 deficiency reduced the plaque area by 58.9% and 37.5% in aorta and aortic sinus, respectively. Plaque instability was aggravated by ALDH2 deficiency along with the increased necrotic core size, decreased collagen content, thinner fibrous cap area, decreased VSMC content, and increased macrophage content. In atherosclerotic lesions, ALDH2 protein was located in both macrophages and VSMCs. Further results revealed downregulated ALDH2 expression in aorta of aged ApoE^?/? mice compared with young mice. However, in vitro study suggested that ALDH2 expression was upregulated in bone marrow-derived macrophages (BMDMs) with an opposite effect in VSMCs following 80 μg/ml oxidized low-density lipoprotein (oxLDL) treatment. Interestingly, ALDH2 deficiency displayed little effect in oxLDL-induced foam cell formation from BMDMs, while ALDH2 knockdown by siRNA and ALDH2 overexpression by lentivirus infection promoted and retarded oxLDL-induced VSMC senescence, respectively. Mechanistically, ALDH2 mitigated oxLDL-induced overproduction of mitochondrial reactive oxygen species (mROS) and activation of downstream p53/p21/p16 pathway. Clearance of mROS by mitoTEMPO significantly reversed the promotive effect of ALDH2 knockdown on VSMC senescence. Taken together, our data revealed that ALDH2 deficiency suppressed atherosclerotic plaque area while facilitating plaque instability possibly through accelerating mROS-mediated VSMC senescence.This article is part of a Special Issue entitled: Genetic and epigenetic regulation of aging and longevity edited by Jun Ren & Megan Yingmei Zhang.     

Chronic Over-Expression of Heat Shock Protein 27 Attenuates Atherogenesis and Enhances Plaque Remodeling: A Combined Histological and Mechanical Assessment of Aortic Lesions

Aims

Expression of Heat Shock Protein-27 (HSP27) is reduced in human coronary atherosclerosis. Over-expression of HSP27 is protective against the early formation of lesions in atherosclerosis-prone apoE^−/− mice (apoE^−/−HSP27^o/e) - however, only in females. We now seek to determine if chronic HSP27 over-expression is protective in a model of advanced atherosclerosis in both male and female apoE^−/− mice.

Methods and Results

After 12 weeks on a high fat diet, serum HSP27 levels rose more than 16-fold in male and female apoE^−/−HSP27^o/e mice, although females had higher levels than males. Relative to apoE^−/− mice, female apoE^−/−HSP27^o/e mice showed reductions in aortic lesion area of 35% for en face and 30% for cross-sectional sinus tissue sections – with the same parameters reduced by 21% and 24% in male cohorts; respectively. Aortic plaques from apoE^−/−HSP27^o/e mice showed almost 50% reductions in the area occupied by cholesterol clefts and free cholesterol, with fewer macrophages and reduced apoptosis but greater intimal smooth muscle cell and collagen content. The analysis of the aortic mechanical properties showed increased vessel stiffness in apoE^−/−HSP27^o/e mice (41% in female, 34% in male) compare to apoE^−/− counterparts.

Conclusions

Chronic over-expression of HSP27 is atheroprotective in both sexes and coincides with reductions in lesion cholesterol accumulation as well as favorable plaque remodeling. These data provide new clues as to how HSP27 may improve not only the composition of atherosclerotic lesions but potentially their stability and resilience to plaque rupture.     

Different attentional dysfunctions in eEF2K−/−, IL1RAPL1−/− and SHANK3Δ11−/− mice

A common feature of several psychiatric disorders is the attentional impairment. eEF2K ^?/?, IL1RAPL1 ^?/? and SHANK3Δ11 ^?/? mice were used as animal models consistently linked to changes in synaptic plasticity, learning and memory. All knockout (KO) mice and their corresponding littermates were submitted to the novel object recognition (NOR) and visual object recognition (VOR) tasks. In the NOR, eEF2K^?/? mice exhibited a normal performance in terms of mean discrimination index, while SHANK3Δ11^?/? and IL1RAPL1 ^?/? mice were impaired when a delay of 2 and 24 hours was introduced. Surprisingly, when submitted to VOR, where the two objects were replaced with two shapes delivered from two iPods, all the mutant mice performed worse than those in the NOR. In VOR, the application of motion to different shapes, to increase attention, improved performance in eEF2K ^?/? and IL1RAPL1 ^?/? but not in SHANK3Δ11 ^?/? mice. In SHANK3Δ11 ^?/? mice, attentional deficit was also present even if different motions were applied to the same shapes or when these mice were repeatedly exposed for 5 days to the context. Behavioral analysis showed that eEF2K^?/? and IL1RAPL1 ^?/? mice had a good flexibility tested in the T‐maze. eEF2K^?/? showed normal self‐grooming. On the basis of previous literature data indicating that SHANK3Δ11 ^?/? showed impaired flexibility and reduced sociability, we identified in this genotype the most exhaustive model showing all the core symptoms of autism spectrum disorder including a heavy visual attention deficit. These findings show the importance of VOR to identify mouse models of autism.     

Dual Benefit of Reduced Cx43 on Atherosclerosis in LDL Receptor-Deficient Mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR^?/?) mice were intercrossed with mice heterozygous for Cx43 (Cx43^+/?mice). Male mice with normal (Cx43^+/+LDLR^?/?) or reduced (Cx43^+/?LDLR^?/?) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43^+/?LDLR^?/?mice compared with Cx43^+/+LDLR^?/?littermate controls. In addition, atheroma in Cx43^+/?LDLR^?/?mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Macrophage autophagy regulates mitochondria‐mediated apoptosis and inhibits necrotic core formation in vulnerable plaques

The vulnerable plaque is a key distinguishing feature of atherosclerotic lesions that can cause acute atherothrombotic vascular disease. This study was designed to explore the effect of autophagy on mitochondria‐mediated macrophage apoptosis and vulnerable plaques. Here, we generated the mouse model of vulnerable carotid plaque in ApoE^?/? mice. Application of ApoE^?/? mice with rapamycin (an autophagy inducer) inhibited necrotic core formation in vulnerable plaques by decreasing macrophage apoptosis. However, 3‐methyladenine (an autophagy inhibitor) promoted plaque vulnerability through deteriorating these indexes. To further explore the mechanism of autophagy on macrophage apoptosis, we used macrophage apoptosis model in vitro and found that 7‐ketocholesterol (7‐KC, one of the primary oxysterols in oxLDL) caused macrophage apoptosis with concomitant impairment of mitochondria, characterized by the impairment of mitochondrial ultrastructure, cytochrome c release, mitochondrial potential dissipation, mitochondrial fragmentation, excessive ROS generation and both caspase‐9 and caspase‐3 activation. Interestingly, such mitochondrial apoptotic responses were ameliorated by autophagy activator, but exacerbated by autophagy inhibitor. Finally, we found that MAPK‐NF‐κB signalling pathway was involved in autophagy modulation of 7‐KC–induced macrophage apoptosis. So, we provide strong evidence for the potential therapeutic benefit of macrophage autophagy in regulating mitochondria‐mediated apoptosis and inhibiting necrotic core formation in vulnerable plaques.     

Ginsenoside Rb1 enhances atherosclerotic plaque stability by skewing macrophages to the M2 phenotype

Atherosclerosis (AS) is characterized as progressive arterial plaque, which is easy to rupture under low stability. Macrophage polarization and inflammation response plays an important role in regulating plaque stability. Ginsenoside Rb1 (Rb1), one of the main active principles of Panax Ginseng, has been found powerful potential in alleviating inflammatory response. However, whether Rb1 could exert protective effects on AS plaque stability remains unclear. This study investigated the role of Rb1 on macrophage polarization and atherosclerotic plaque stability using primary peritoneal macrophages isolated from C57BL/6 mice and AS model in ApoE^?/? mice. In vitro, Rb1 treatment promoted the expression of arginase‐I (Arg‐I) and macrophage mannose receptor (CD206), two classic M2 macrophages markers, while the expression of iNOS (M1 macrophages) was decreased. Rb1 increased interleukin‐4 (IL‐4) and interleukin‐13 (IL‐13) secretion in supernatant and promoted STAT6 phosphorylation. IL‐4 and/or IL‐13 neutralizing antibodies and leflunomide, a STAT6 inhibitor attenuated the up‐regulation of M2 markers induced by Rb1. In vivo, the administration of Rb1 promoted atherosclerotic lesion stability, accompanied by increased M2 macrophage phenotype and reduced MMP‐9 staining. These data suggested that Rb1 enhanced atherosclerotic plaque stability through promoting anti‐inflammatory M2 macrophage polarization, which is achieved partly by increasing the production of IL‐4 and/or IL‐13 and STAT6 phosphorylation. Our study provides new evidence for possibility of Rb1 in prevention and treatment of atherosclerosis.     

MDA‐5 activation by cytoplasmic double‐stranded RNA impairs endothelial function and aggravates atherosclerosis

Recent studies have highlighted the relevance of viral nucleic acid immunorecognition by pattern recognition receptors in atherogenesis. Melanoma differentiation associated gene 5 (MDA‐5) belongs to the intracellular retinoic acid inducible gene‐I like receptors and its activation promotes pro‐inflammatory mechanisms. Here, we studied the effect of MDA‐5 stimulation in vascular biology. To gain insights into MDA‐5 dependent effects on endothelial function, cultured human coronary artery endothelial cells (HCAEC) were transfected with the synthetic MDA‐5 agonist polyIC (long double‐stranded RNA). Human coronary endothelial cell expressed MDA‐5 and reacted with receptor up‐regulation upon stimulation. Reactive oxygen species formation, apoptosis and the release of pro‐inflammatory cytokines was enhanced, whereas migration was significantly reduced in response to MDA‐5 stimulation. To test these effects in vivo, wild‐type mice were transfected with 32.5 μg polyIC/JetPEI or polyA/JetPEI as control every other day for 7 days. In polyIC‐treated wild‐type mice, endothelium‐dependent vasodilation and re‐endothelialization was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticles and circulating endothelial progenitor cells significantly elevated compared to controls. Importantly, these effects could be abrogated by MDA‐5 deficiency in vivo. Finally, chronic MDA‐5 stimulation in Apolipoprotein E/toll‐like receptor 3 (TLR3) double^‐deficient (ApoE^?/?/TLR3^?/?) mice‐enhanced atherosclerotic plaque formation. This study demonstrates that MDA‐5 stimulation leads to endothelial dysfunction, and has the potential to aggravate atherosclerotic plaque burden in murine atherosclerosis. Thus, the spectrum of relevant innate immune receptors in vascular diseases and atherogenesis might not be restricted to TLRs but also encompasses the group of RLRs including MDA‐5.