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.     

Dihydrocapsaicin Attenuates Plaque Formation through a PPARγ/LXRα Pathway in apoE?/? Mice Fed a High-Fat/High-Cholesterol Diet

Aims

Atherosclerosis is a chronic inflammatory disease and represents the major cause of cardiovascular morbidity and mortality. There is evidence that dihydrocapsaicin (DHC) can exert multiple pharmacological and physiological effects. Here, we explored the effect of DHC in atherosclerotic plaque progression in apoE^−/− mice fed a high-fat/high-cholesterol diet.

Methods and Results

apoE^−/− mice were randomly divided into two groups and fed a high-fat/high-cholesterol diet with or without DHC for 12 weeks. We demonstrated that cellular cholesterol content was significantly decreased while apoA1-mediated cholesterol efflux was significantly increased following treatment with DHC in THP-1 macrophage-derived foam cells. We also observed that plasma levels of TG, LDL-C, VLDL-C, IL-1β, IL-6, TNF-α and CRP were markedly decreased while plasma levels of apoA1 and HDL-C were significantly increased, and consistent with this, atherosclerotic lesion development was significantly inhibited by DHC treatment of apoE^−/− mice fed a high-fat/high-cholesterol diet. Moreover, treatment with both LXRα siRNA and PPARγ siRNA made the up-regulation of DHC on ABCA1, ABCG1, ABCG5, SR-B1, NPC1, CD36, LDLR, HMGCR, apoA1 and apoE expression notably abolished while made the down-regulation of DHC on SRA1 expression markedly compensated. And treatment with PPARγ siRNA made the DHC-induced up-regulation of LXRα expression notably abolished while treatment with LXRα siRNA had no effect on DHC-induced PPARγ expression.

Conclusion

These observations provide direct evidence that DHC can significantly decrease atherosclerotic plaque formation involving in a PPARγ/LXRα pathway and thus DHC may represent a promising candidate for a therapeutic agent for the treatment or prevention of atherosclerosis.     

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.     

MicroRNA-590 Inhibits Lipoprotein Lipase Expression and Prevents Atherosclerosis in apoE Knockout Mice

Recent studies have suggested that miR-590 may play critical roles in cardiovascular disease. This study was designed to determine the effects of miR-590 on lipoprotein lipase (LPL) expression and development of atherosclerosis in apolipoprotein E knockout (apoE^−/−) mice and explore the potential mechanisms. En face analysis of the whole aorta revealed that miR-590 significantly decreased aortic atherosclerotic plaque size and lipid content in apoE^−/− mice. Double immunofluorescence staining in cross-sections of the proximal aorta showed that miR-590 agomir reduced CD68 and LPL expression in macrophages in atherosclerotic lesions. MiR-590 agomir down-regulated LPL mRNA and protein expression as analyzed by RT-qPCR and western blotting analyses, respectively. Consistently, miR-590 decreased the expression of CD36 and scavenger receptor A1 (SRA1) mRNA and protein. High-performance liquid chromatography (HPLC)analysis confirmed that treatment with miR-590 agomir reduced lipid levels either in plasma orinabdominal cavity macrophages of apoE^−/− mice. ELISA analysis showed that miR-590 agomir decreased plasma levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukin-1β (IL-1β)and interleukin-6 (IL-6). In contrast, treatment with miR-590 antagomir prevented or reversed these effects. Taken together, these results reveal a novel mechanism of miR-590 effects, and may provide new insights into the development of strategies for attenuating lipid accumulation and pro-inflammatory cytokine secretion.     

Celastrol Prevents Atherosclerosis via Inhibiting LOX-1 and Oxidative Stress

Celastrol is a triterpenoid compound extracted from the Chinese herb Tripterygium wilfordii Hook F. Previous research has revealed its anti-oxidant, anti-inflammatory, anti-cancer and immunosuppressive properties. Here, we investigated whether celastrol inhibits oxidized low-density lipoprotein (oxLDL) induced oxidative stress in RAW 264.7 cells. In addition, the effect of celastrol on atherosclerosis in vivo was assessed in apolipoprotein E knockout (apoE^−/−) mouse fed a high-fat/high-cholesterol diet (HFC). We found that celastrol significantly attenuated oxLDL-induced excessive expression of lectin-like oxidized low density lipoprotein receptor-1(LOX-1) and generation of reactive oxygen species (ROS) in cultured RAW264.7 macrophages. Celastrol also decreased IκB phosphorylation and degradation and reduced production of inducible nitric oxide synthase (iNOS), nitric oxide (NO) and proinflammatory cytokines such as tumor necrosis factor (TNF)-α and IL-6. Celastrol reduced atherosclerotic plaque size in apoE^−/− mice. The expression of LOX-1 within the atherosclerotic lesions and generation of superoxide in mouse aorta were also significantly reduced by celastrol while the lipid profile was not improved. In conclusion, our results show that celastrol inhibits atherosclerotic plaque developing in apoE^−/− mice via inhibiting LOX-1 and oxidative stress.     

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.     

A lincRNA-DYNLRB2-2/GPR119/GLP-1R/ABCA1-dependent signal transduction pathway is essential for the regulation of cholesterol homeostasis

Accumulated evidence shows that G protein-coupled receptor 119 (GPR119) plays a key role in glucose and lipid metabolism. Here, we explored the effect of GPR119 on cholesterol metabolism and inflammation in THP-1 macrophages and atherosclerotic plaque progression in apoE^−/− mice. We found that oxidized LDL (Ox-LDL) significantly induced long intervening noncoding RNA (lincRNA)-DYNLRB2-2 expression, resulting in the upregulation of GPR119 and ABCA1 expression through the glucagon-like peptide 1 receptor signaling pathway. GPR119 significantly decreased cellular cholesterol content and increased apoA-I-mediated cholesterol efflux in THP-1 macrophage-derived foam cells. In vivo, apoE^−/− mice were randomly divided into two groups and infected with lentivirus (LV)-Mock or LV-GPR119 for 8 weeks. GPR119-treated mice showed decreased liver lipid content and plasma TG, interleukin (IL)-1β, IL-6, and TNF-α levels, whereas plasma levels of apoA-I were significantly increased. Consistent with this, atherosclerotic lesion development was significantly inhibited by infection of apoE^−/− mice with LV-GPR119. Our findings clearly indicate that, Ox-LDL significantly induced lincRNA-DYNLRB2-2 expression, which promoted ABCA1-mediated cholesterol efflux and inhibited inflammation through GPR119 in THP-1 macrophage-derived foam cells. Moreover, GPR119 decreased lipid and serum inflammatory cytokine levels, decreasing atherosclerosis in apoE^−/− mice. These suggest that GPR119 may be a promising candidate as a therapeutic agent.     

t-10, c-12 CLA Dietary Supplementation Inhibits Atherosclerotic Lesion Development Despite Adverse Cardiovascular and Hepatic Metabolic Marker Profiles

Animal and human studies have indicated that fatty acids such as the conjugated linoleic acids (CLA) found in milk could potentially alter the risk of developing metabolic disorders including diabetes and cardiovascular disease (CVD). Using susceptible rodent models (apoE^−/− and LDLr^−/− mice) we investigated the interrelationship between mouse strain, dietary conjugated linoleic acids and metabolic markers of CVD. Despite an adverse metabolic risk profile, atherosclerosis (measured directly by lesion area), was significantly reduced with t-10, c-12 CLA and mixed isomer CLA (Mix) supplementation in both apoE^−/− (p<0.05, n = 11) and LDLr^−/− mice (p<0.01, n = 10). Principal component analysis was utilized to delineate the influence of multiple plasma and tissue metabolites on the development of atherosclerosis. Group clustering by dietary supplementation was evident, with the t-10, c-12 CLA supplemented animals having distinct patterns, suggestive of hepatic insulin resistance, regardless of mouse strain. The effect of CLA supplementation on hepatic lipid and fatty acid composition was explored in the LDLr^−/− strain. Dietary supplementation with t-10, c-12 CLA significantly increased liver weight (p<0.05, n = 10), triglyceride (p<0.01, n = 10) and cholesterol ester content (p<0.01, n = 10). Furthermore, t-10, c-12 CLA also increased the ratio of 18∶1 to 18∶0 fatty acid in the liver suggesting an increase in the activity of stearoyl-CoA desaturase. Changes in plasma adiponectin and liver weight with t-10, c-12 CLA supplementation were evident within 3 weeks of initiation of the diet. These observations provide evidence that the individual CLA isomers have divergent mechanisms of action and that t-10, c-12 CLA rapidly changes plasma and liver markers of metabolic syndrome, despite evidence of reduction in atherosclerosis.     

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.     

Nicotinic acid and DP1 blockade: studies in mouse models of atherosclerosis

The use of nicotinic acid to treat dyslipidemia is limited by induction of a “flushing” response, mediated in part by the interaction of prostaglandin D₂ (PGD₂) with its G-protein coupled receptor, DP1 (Ptgdr). The impact of DP1 blockade (genetic or pharmacologic) was assessed in experimental murine models of atherosclerosis. In Ptgdr^−/−ApoE^−/− mice versus ApoE^−/− mice, both fed a high-fat diet, aortic cholesterol content was modestly higher (1.3- to 1.5-fold, P < 0.05) in Ptgdr^−/−ApoE^−/− mice at 16 and 24 weeks of age, but not at 32 weeks. In multiple ApoE^−/− mouse studies, a DP1-specific antagonist, L-655, generally had a neutral to beneficial effect on aortic lipids in the presence or absence of nicotinic acid treatment. In a separate study, a modest increase in some atherosclerotic measures was observed with L-655 treatment in Ldlr^−/− mice fed a high-fat diet for 8 weeks; however, this effect was not sustained for 16 or 24 weeks. In the same study, treatment with nicotinic acid alone generally decreased plasma and/or aortic lipids, and addition of L-655 did not negate those beneficial effects. These studies demonstrate that inhibition of DP1, with or without nicotinic acid treatment, does not lead to consistent or sustained effects on plaque burden in mouse atherosclerotic models.     

Nur77 Decreases Atherosclerosis Progression in apoE?/? Mice Fed a High-Fat/High-Cholesterol Diet

Rationale

It is clear that lipid disorder and inflammation are associated with cardiovascular diseases and underlying atherosclerosis. Nur77 has been shown to be involved in inflammatory response and lipid metabolism.

Objective

Here, we explored the role of Nur77 in atherosclerotic plaque progression in apoE^−/− mice fed a high-fat/high cholesterol diet.

Methods and Results

The Nur77 gene, a nuclear hormone receptor, was highly induced by treatment with Cytosporone B (Csn-B, specific Nur77 agonist), recombinant plasmid over-expressing Nur77 (pcDNA-Nur77), while inhibited by treatment with siRNAs against Nur77 (si-Nur77) in THP-1 macrophage-derived foam cells, HepG2 cells and Caco-2 cells, respectively. In addition, the expression of Nur77 was highly induced by Nur77 agonist Csn-B, lentivirus encoding Nur77 (LV-Nur77), while silenced by lentivirus encoding siRNA against Nur77 (si-Nur77) in apoE^−/− mice fed a high-fat/high cholesterol diet, respectively. We found that increased expression of Nur77 reduced macrophage-derived foam cells formation and hepatic lipid deposition, downregulated gene levels of inflammatory molecules, adhesion molecules and intestinal lipid absorption, and decreases atherosclerotic plaque formation.

Conclusion

These observations provide direct evidence that Nur77 is an important nuclear hormone receptor in regulation of atherosclerotic plaque formation and thus represents a promising target for the treatment of atherosclerosis.     

Self-Gated CINE MRI for Combined Contrast-Enhanced Imaging and Wall-Stiffness Measurements of Murine Aortic Atherosclerotic Lesions

Background

High-resolution contrast-enhanced imaging of the murine atherosclerotic vessel wall is difficult due to unpredictable flow artifacts, motion of the thin artery wall and problems with flow suppression in the presence of a circulating contrast agent.

Methods and Results

We applied a 2D-FLASH retrospective-gated CINE MRI method at 9.4T to characterize atherosclerotic plaques and vessel wall distensibility in the aortic arch of aged ApoE^−/− mice after injection of a contrast agent. The method enabled detection of contrast enhancement in atherosclerotic plaques in the aortic arch after I.V. injection of micelles and iron oxides resulting in reproducible plaque enhancement. Both contrast agents were taken up in the plaque, which was confirmed by histology. Additionally, the retrospective-gated CINE method provided images of the aortic wall throughout the cardiac cycle, from which the vessel wall distensibility could be calculated. Reduction in plaque size by statin treatment resulted in lower contrast enhancement and reduced wall stiffness.

Conclusions

The retrospective-gated CINE MRI provides a robust and simple way to detect and quantify contrast enhancement in atherosclerotic plaques in the aortic wall of ApoE^−/− mice. From the same scan, plaque-related changes in stiffness of the aortic wall can be determined. In this mouse model, a correlation between vessel wall stiffness and atherosclerotic lesions was found.     

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.     

Hepatic overexpression of methionine sulfoxide reductase A reduces atherosclerosis in apolipoprotein E-deficient mice

Methionine sulfoxide reductase A (MsrA), a specific enzyme that converts methionine-S-sulfoxide to methionine, plays an important role in the regulation of protein function and the maintenance of redox homeostasis. In this study, we examined the impact of hepatic MsrA overexpression on lipid metabolism and atherosclerosis in apoE-deficient (apoE^−/−) mice. In vitro study showed that in HepG2 cells, lentivirus-mediated human MsrA (hMsrA) overexpression upregulated the expression levels of several key lipoprotein-metabolism-related genes such as liver X receptor α, scavenger receptor class B type I, and ABCA1. ApoE^−/− mice were intravenously injected with lentivirus to achieve high-level hMsrA expression predominantly in the liver. We found that hepatic hMsrA expression significantly reduced plasma VLDL/LDL levels, improved plasma superoxide dismutase, and paraoxonase-1 activities, and decreased plasma serum amyloid A level in apoE^−/− mice fed a Western diet, by significantly altering the expression of several genes in the liver involving cholesterol selective uptake, conversion and excretion into bile, TG biosynthesis, and inflammation. Moreover, overexpression of hMsrA resulted in reduced hepatic steatosis and aortic atherosclerosis. These results suggest that hepatic MsrA may be an effective therapeutic target for ameliorating dyslipidemia and reducing atherosclerosis-related cardiovascular diseases.     

An Immunomodulating Fatty Acid Analogue Targeting Mitochondria Exerts Anti-Atherosclerotic Effect beyond Plasma Cholesterol-Lowering Activity in apoE-/- Mice

Tetradecylthioacetic acid (TTA) is a hypolipidemic antioxidant with immunomodulating properties involving activation of peroxisome proliferator-activated receptors (PPARs) and proliferation of mitochondria. This study aimed to penetrate the effect of TTA on the development of atherosclerotic lesions in apolipoprotein (apo)-E^-/- mice fed a high-fat diet containing 0.3% TTA for 12 weeks. These mice displayed a significantly less atherosclerotic development vs control. Plasma cholesterol was increased by TTA administration and triacylglycerol (TAG) levels in plasma and liver were decreased by TTA supplementation, the latter, probably due to increased mitochondrial fatty acid oxidation and reduced lipogenesis. TTA administration also changed the fatty acid composition in the heart, and the amount of arachidonic acid (ARA) and eicosapentaenoic acid (EPA) was reduced and increased, respectively. The heart mRNA expression of inducible nitric oxidase (NOS)-2 was decreased in TTA-treated mice, whereas the mRNA level of catalase was increased. Finally, reduced plasma levels of inflammatory mediators as IL-1α, IL-6, IL-17, TNF-α and IFN-γ were detected in TTA-treated mice. These data show that TTA reduces atherosclerosis in apoE^-/- mice and modulates risk factors related to atherosclerotic disorders. TTA probably acts at both systemic and vascular levels in a manner independent of changes in plasma cholesterol, and triggers TAG catabolism through improved mitochondrial function.     

CTRP12 ameliorates atherosclerosis by promoting cholesterol efflux and inhibiting inflammatory response via the miR-155-5p/LXRα pathway

C1q tumor necrosis factor-related protein 12 (CTRP12), a conserved paralog of adiponectin, is closely associated with cardiovascular disease. However, little is known about its role in atherogenesis. The aim of this study was to examine the influence of CTRP12 on atherosclerosis and explore the underlying mechanisms. Our results showed that lentivirus-mediated CTRP12 overexpression inhibited lipid accumulation and inflammatory response in lipid-laden macrophages. Mechanistically, CTRP12 decreased miR-155-5p levels and then increased its target gene liver X receptor α (LXRα) expression, which increased ATP binding cassette transporter A1 (ABCA1)- and ABCG1-dependent cholesterol efflux and promoted macrophage polarization to the M2 phenotype. Injection of lentiviral vector expressing CTRP12 decreased atherosclerotic lesion area, elevated plasma high-density lipoprotein cholesterol levels, promoted reverse cholesterol transport (RCT), and alleviated inflammatory response in apolipoprotein E-deficient (apoE^−/−) mice fed a Western diet. Similar to the findings of in vitro experiments, CTRP12 overexpression diminished miR-155-5p levels but increased LXRα, ABCA1, and ABCG1 expression in the aortas of apoE^−/− mice. Taken together, these results suggest that CTRP12 protects against atherosclerosis by enhancing RCT efficiency and mitigating vascular inflammation via the miR-155-5p/LXRα pathway. Stimulating CTRP12 production could be a novel approach for reducing atherosclerosis.Subject terms: Non-coding RNAs, Cardiovascular diseases     

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.     

Serum amyloid A augments the atherogenic effects of cholesteryl ester transfer protein

Serum amyloid A (SAA) is predictive of CVD in humans and causes atherosclerosis in mice. SAA has many proatherogenic effects in vitro. However, HDL, the major carrier of SAA in the circulation, masks these effects. The remodeling of HDL by cholesteryl ester transfer protein (CETP) liberates SAA restoring its proinflammatory activity. Here, we investigated whether deficiency of SAA suppresses the previously described proatherogenic effect of CETP. ApoE^−/− mice and apoE^−/− mice deficient in the three acute-phase isoforms of SAA (SAA1.1, SAA2.1, and SAA3; “apoE^−/− SAA-TKO”) with and without adeno-associated virus-mediated expression of CETP were studied. There was no effect of CETP expression or SAA genotype on plasma lipids or inflammatory markers. Atherosclerotic lesion area in the aortic arch of apoE^−/− mice was 5.9 ± 1.2%; CETP expression significantly increased atherosclerosis in apoE^−/− mice (13.1 ± 2.2%). However, atherosclerotic lesion area in the aortic arch of apoE^−/− SAA-TKO mice (5.1 ± 1.1%) was not significantly increased by CETP expression (6.2 ± 0.9%). The increased atherosclerosis in apoE^−/− mice expressing CETP was associated with markedly increased SAA immunostaining in aortic root sections. Thus, SAA augments the atherogenic effects of CETP, which suggests that inhibiting CETP may be of particular benefit in patients with high SAA.     

Raised Soluble P-Selectin Moderately Accelerates Atherosclerotic Plaque Progression

Soluble P-selectin (sP-selectin), a biomarker of inflammatory related pathologies including cardiovascular and peripheral vascular diseases, also has pro-atherosclerotic effects including the ability to increase leukocyte recruitment and modulate thrombotic responses in vivo. The current study explores its role in progressing atherosclerotic plaque disease. Apoe ^−/− mice placed on a high fat diet (HFD) were given daily injections of recombinant dimeric murine P-selectin (22.5 µg/kg/day) for 8 or 16 weeks. Saline or sE-selectin injections were used as negative controls. In order to assess the role of sP-selectin on atherothrombosis an experimental plaque remodelling murine model, with sm22α-hDTR Apoe^−/− mice on a HFD in conjunction with delivery of diphtheria toxin to induce targeted vascular smooth muscle apoptosis, was used. These mice were similarly given daily injections of sP-selectin for 8 or 16 weeks. While plaque mass and aortic lipid content did not change with sP-selectin treatment in Apoe^−/− or SM22α-hDTR Apoe^−/− mice on HFD, increased plasma MCP-1 and a higher plaque CD45 content in Apoe ^−/− HFD mice was observed. As well, a significant shift towards a more unstable plaque phenotype in the SM22α-hDTR Apoe^−/− HFD mice, with increased macrophage accumulation and lower collagen content, leading to a lower plaque stability index, was observed. These results demonstrate that chronically raised sP-selectin favours progression of an unstable atherosclerotic plaque phenotype.