The Modulation of Endoplasmic Reticulum Stress by Chemical Chaperone Upregulates Immune Negative Cytokine IL-35 in Apolipoprotein E-Deficient Mice

Interleukin (IL)-35 is a newly identified immune negative molecule which is secreted by CD4⁺Foxp3⁺ T regulatory cells (Tregs) and contributes to their suppressive capacity. Early data have shown that IL-35 inhibits development of several autoimmune diseases. However, the role of IL-35 in atherosclerosis, a lipid-driven chronic inflammatory disease in arterial wall, remains to be investigated. Here, we found that IL-35 was involved in atherosclerosis in apolipoprotein E-deficient (ApoE^−/−) mice. ApoE^−/− mice with established atherosclerotic lesion displayed a lower level of IL-35 compared to age-matched wild type C57BL/6 mice without plaque. However, IL-35 expression increased significantly in ApoE^−/− mice with attenuated plaque. More importantly, we found that modulation of ER stress treated by chemical chaperone, 4-Phenyl butyric acid (PBA) in vivo, mainly upregulated immune negative regulating molecule IL-35, as well as IL-10 and Foxp3, accompanied by increased Tregs. However, no obvious impact on pro-inflammatory molecules such as TNF-α, IFN-γ, IL-17 and IL-23 was observed, which provides new insight into the benefit of ER stress recovery from attenuated plaque. Our results suggest that IL-35 might have a potential value for atherosclerotic therapy.     

Irgm 1参与动脉粥样硬化斑块的形成

目的:探讨免疫相关GTP酶1(Irgm 1)对小鼠血管动脉粥样硬化(AS)斑块形成的影响。方法:高脂饲料喂养野生型(WT)、ApoE~(-/-)Irgm 1~(+/+)和ApoE~(-/-)Irgm1~(+/-)小鼠3个月,建立AS模型;取小鼠主动脉弓,免疫荧光染色方法观察WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1的表达情况及部位;Western blot方法检测WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1蛋白表达情况;Q-PCR方法检测WT和ApoE~(-/-)Irgm 1~(+/+)小鼠血管AS斑块中Irgm 1 m RNA表达情况;油红O染色观察ApoE~(-/-)Irgm1~(+/+)和ApoE~(-/-)Irgm1~(+/-)小鼠血管AS斑块形成情况;结果:与WT组相比,ApoE~(-/-)Irgm 1~(+/+)组小鼠主动脉弓AS斑块中Irgm 1+细胞明显增多,Irgm 1+细胞主要位于血管AS斑块的表面;与WT组相比,ApoE~(-/-)Irgm 1~(+/+)组小鼠血管AS斑块中Irgm 1蛋白表达显著增多(P0.001),Irgm 1 m RNA表达显著增多(P0.01);与ApoE~(-/-)Irgm1~(+/-)组相比,ApoE~(-/-)Irgm1~(+/+)组小鼠主动脉弓AS斑块面积显著增大(P0.01);结论:Irgm 1能够促进血管AS斑块的形成。     

Adiponectin Receptor 2 Deficiency Results in Reduced Atherosclerosis in the Brachiocephalic Artery in Apolipoprotein E Deficient Mice

Adiponectin has been shown to have beneficial cardiovascular effects and to signal through the adiponectin receptors, AdipoR1 and AdipoR2. The original aim of this study was to investigate the effect of combined AdipoR1 and AdipoR2 deficiency (AdipoR1^-/-AdipoR2^-/-) on atherosclerosis. However, we made the interesting observation that AdipoR1 ^-/- AdipoR2 ^-/- leads to embryonic lethality demonstrating the critical importance of the adiponectin signalling system during development. We then investigated the effect of AdipoR2-ablation on the progression of atherosclerosis in apolipoprotein E deficient (ApoE ^-/-) mice. AdipoR2^-/-ApoE^-/- mice fed an atherogenic diet had decreased plaque area in the brachiocephalic artery compared with AdipoR2 ^+/+ApoE^-/- littermate controls as visualized in vivo using an ultrasound biomicroscope and confirmed by histological analyses. The decreased plaque area in the brachiocephalic artery could not be explained by plasma cholesterol levels or inflammatory status. However, accumulation of neutral lipids was decreased in peritoneal macrophages from AdipoR2^-/-ApoE^-/- mice after incubation with oxidized LDL. This effect was associated with lower CD36 and higher ABCA1 mRNA levels in peritoneal macrophages from AdipoR2^-/-ApoE^-/- mice compared with AdipoR2^+/+ApoE^-/- controls after incubation with oxidized LDL. In summary, we show that adiponectin receptors are crucial during embryonic development and that AdipoR2-deficiency slows down the progression of atherosclerosis in the brachiocephalic artery of ApoE-deficient mice.     

Dual Activation of the Bile Acid Nuclear Receptor FXR and G-Protein-Coupled Receptor TGR5 Protects Mice against Atherosclerosis

Bile acid signaling is a critical regulator of glucose and energy metabolism, mainly through the nuclear receptor FXR and the G protein-coupled receptor TGR. The purpose of the present study was to investigate whether dual activation of FXR and TGR5 plays a significant role in the prevention of atherosclerosis progression. To evaluate the effects of bile acid signaling in atherogenesis, ApoE^−/− mice and LDLR^−/− mice were treated with an FXR/TGR5 dual agonist (INT-767). INT-767 treatment drastically reduced serum cholesterol levels. INT-767 treatment significantly reduced atherosclerotic plaque formation in both ApoE^−/− and LDLR^−/− mice. INT-767 decreased the expression of pro-inflammatory cytokines and chemokines in the aortas of ApoE^−/− mice through the inactivation of NF-κB. In addition, J774 macrophages treated with INT-767 had significantly lower levels of active NF-κB, resulting in cytokine production in response to LPS through a PKA dependent mechanism. This study demonstrates that concurrent activation of FXR and TGR5 attenuates atherosclerosis by reducing both circulating lipids and inflammation.     

Polymicrobial Oral Infection with Four Periodontal Bacteria Orchestrates a Distinct Inflammatory Response and Atherosclerosis in ApoEnull Mice

Periodontal disease (PD) develops from a synergy of complex subgingival oral microbiome, and is linked to systemic inflammatory atherosclerotic vascular disease (ASVD). To investigate how a polybacterial microbiome infection influences atherosclerotic plaque progression, we infected the oral cavity of ApoE^null mice with a polybacterial consortium of 4 well-characterized periodontal pathogens, Porphyromonas gingivalis, Treponema denticola, Tannerealla forsythia and Fusobacterium nucleatum, that have been identified in human atherosclerotic plaque by DNA screening. We assessed periodontal disease characteristics, hematogenous dissemination of bacteria, peripheral T cell response, serum inflammatory cytokines, atherosclerosis risk factors, atherosclerotic plaque development, and alteration of aortic gene expression. Polybacterial infections have established gingival colonization in ApoE^null hyperlipidemic mice and displayed invasive characteristics with hematogenous dissemination into cardiovascular tissues such as the heart and aorta. Polybacterial infection induced significantly higher levels of serum risk factors oxidized LDL (p < 0.05), nitric oxide (p < 0.01), altered lipid profiles (cholesterol, triglycerides, Chylomicrons, VLDL) (p < 0.05) as well as accelerated aortic plaque formation in ApoE^null mice (p < 0.05). Periodontal microbiome infection is associated with significant decreases in Apoa1, Apob, Birc3, Fga, FgB genes that are associated with atherosclerosis. Periodontal infection for 12 weeks had modified levels of inflammatory molecules, with decreased Fas ligand, IL-13, SDF-1 and increased chemokine RANTES. In contrast, 24 weeks of infection induced new changes in other inflammatory molecules with reduced KC, MCSF, enhancing GM-CSF, IFNγ, IL-1β, IL-13, IL-4, IL-13, lymphotactin, RANTES, and also an increase in select inflammatory molecules. This study demonstrates unique differences in the host immune response to a polybacterial periodontal infection with atherosclerotic lesion progression in a mouse model.     

Chronic Internal Exposure to Low Dose 137Cs Induces Positive Impact on the Stability of Atherosclerotic Plaques by Reducing Inflammation in ApoE-/- Mice

After Chernobyl and Fukushima Daï Chi, two major nuclear accidents, large amounts of radionuclides were released in the environment, mostly caesium 137 (¹³⁷Cs). Populations living in contaminated territories are chronically exposed to radionuclides by ingestion of contaminated food. However, questions still remain regarding the effects of low dose ionizing radiation exposure on the development and progression of cardiovascular diseases. We therefore investigated the effects of a chronic internal exposure to ¹³⁷Cs on atherosclerosis in predisposed ApoE^-/- mice. Mice were exposed daily to 0, 4, 20 or 100 kBq/l ¹³⁷Cs in drinking water, corresponding to range of concentrations found in contaminated territories, for 6 or 9 months. We evaluated plaque size and phenotype, inflammatory profile, and oxidative stress status in different experimental groups. Results did not show any differences in atherosclerosis progression between mice exposed to ¹³⁷Cs and unexposed controls. However, ¹³⁷Cs exposed mice developed more stable plaques with decreased macrophage content, associated with reduced aortic expression of pro-inflammatory factors (CRP, TNFα, MCP-1, IFNγ) and adhesion molecules (ICAM-1, VCAM-1 and E-selectin). Lesions of mice exposed to ¹³⁷Cs were also characterized by enhanced collagen and smooth muscle cell content, concurrent with reduced matrix metalloproteinase MMP8 and MMP13 expression. These results suggest that low dose chronic exposure of ¹³⁷Cs in ApoE^-/- mice enhances atherosclerotic lesion stability by inhibiting pro-inflammatory cytokine and MMP production, resulting in collagen-rich plaques with greater smooth muscle cell and less macrophage content.     

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.     

Effects of emotional and physiological stress on plaque instability in apolipoprotein E knockout mice

In the present study, we sought to investigate the effects of emotional and physiological stress on plaque instability in atherosclerosis. We used different stress-treated apolipoprotein E (ApoE)-deficient mice, which have been shown to spontaneously develop atherosclerosis with features similar to those seen in humans, as an animal model. Morphology study showed that emotional stress (ES) obviously promoted the development of atherosclerotic plaques and plaque instability evidenced by significantly increasing plaque size, plaque-to-surface ratio and plaque calcification, and enhancing the frequency of large necrotic core and medial erosion compared with control ApoE^−/− mice (P < 0.01). Physiological stress (PS) treatment alone did not affect the plaque stability compared with control ApoE^−/− mice (P > 0.05). However, the combination of ES and PS treatment (CS) initiated much stronger plaque instability compared with ES treatment alone (P < 0.01), increased the frequency of thin fibrous caps, and even triggered plaque rupture and buried fibrous cap. Immunohistochemical analysis indicated that both ES and CS treatment led to an increase in the accumulation of macrophages and T cells and a decrease of smooth muscle cells, reflecting an unstable atherosclerotic plaque phenotype, in the atherosclerotic lesions in ApoE^−/− mice. PS alone did not affect plaque cellular components. Similarly, CS-mediated changes in atherosclerotic plaque composition were stronger than that caused by ES alone (P < 0.01). Taken together, ES treatment alone is sufficient to promote plaque instability. PS alone does not affect atherosclerotic plaque development, but can potentiate ES-mediated plaque destabilization.     

Butanol Extract of Acanthopanax senticosus (Rupr. et Maxim.) Harms Alleviates Atherosclerosis in Apolipoprotein E-Deficient Mice Fed a High-Fat Diet

This study investigated the effect of butanol extract of AS (ASBUE) on atherosclerosis in apolipoprotein E-deficient (ApoE^−/−) mice. The mice were administered ASBUE (390 or 130 mg/kg/day) or rosuvastatin (RSV) via oral gavage for eight weeks. In ApoE^−/− mice, ASBUE suppressed the abnormal body weight gain and improved serum and liver biochemical indicators. ASBUE remarkably reduced the aortic plaque area, improved liver pathological conditions, and lipid metabolism abnormalities, and altered the intestinal microbiota structure in ApoE^−/− mice. In the vascular tissue of ASBUE-treated mice, P-IKKβ, P-NFκB, and P-IκBα levels tended to decrease, while IκB-α increased in high fat-diet-fed atherosclerotic mice. These findings demonstrated the anti-atherosclerotic potential of ASBUE, which is mediated by the interaction between the gut microbiota and lipid metabolism and regulated via the Nuclear Factor-kappa B (NF-κB) pathway. This work paves the groundwork for subsequent studies to develop innovative drugs to treat atherosclerosis.     

Citreoviridin Enhances Atherogenesis in Hypercholesterolemic ApoE-Deficient Mice via Upregulating Inflammation and Endothelial Dysfunction

Vascular endothelial dysfunction and inflammatory response are early events during initiation and progression of atherosclerosis. In vitro studies have described that CIT markedly upregulates expressions of ICAM-1 and VCAM-1 of endothelial cells, which result from NF-κB activation induced by CIT. In order to determine whether it plays a role in atherogenesis in vivo, we conducted the study to investigate the effects of CIT on atherosclerotic plaque development and inflammatory response in apolipoprotein E deficient (apoE^-/-) mice. Five-week-old apoE^-/- mice were fed high-fat diets and treated with CIT for 15 weeks, followed by assay of atherosclerotic lesions. Nitric oxide (NO), vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) were detected in serum. Levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), VEGF, and ET-1 in plaque areas of artery walls were examined. NF-κB p65 expression and NF-κB activation in aorta also were assessed. CIT treatment significantly augmented atherosclerotic plaques and increased expressions of ICAM-1, VCAM-1, VEGF and ET-1 in aorta. Mechanistic studies showed that activation of NF-κB was significantly elevated by CIT treatment, indicating the effect of CIT on atherosclerosis may be regulated by activation of NF-κB.     

Transgenic CGI-58 expression in macrophages alleviates the atherosclerotic lesion development in ApoE knockout mice

Comparative Gene Identification-58 (CGI-58), as an adipose triglyceride lipase (ATGL) activator, strongly increases ATGL-mediated triglyceride (TG) catabolism. Previous studies have shown that CGI-58 affects intestinal cholesterol homeostasis independently of ATGL activity. Therefore, we hypothesized that CGI-58 was involved in macrophage cholesterol metabolism and consequently atherosclerotic lesion formation. Here, we generated macrophage-specific CGI-58 transgenic mice (Mac-CGI-58 Tg) using an SRA promoter, which was further mated with ApoE^−/− mice to create litters of CGI-58 Tg/ApoE^−/− mice. These CGI-58 Tg/ApoE^−/− mice exhibited an anti-atherosclerosis phenotype compared with wild type (WT) controls (CGI-58 WT/ApoE^−/−), illustrated by less plaque area in aortic roots. Moreover, macrophage-specific CGI-58 overexpression in mice resulted in up-regulated levels of plasma total cholesterol and HDL-cholesterol. Consequently, higher expression levels of PPARa, PPARγ, LXRα, ABCA1, and ABCG1 were detected in macrophages from CGI-58 Tg/ApoE^−/− mice compared to CGI-58 WT/ApoE^−/− counterparts, which were accompanied by elevated macrophage cholesterol efflux toward HDL and Apo A1. Nevertheless, serum levels of TNF-α and IL-6 were reduced by macrophage-specific CGI-58 overexpression. Finally, bone marrow (BM) transplantation experiments further revealed that ApoE^−/− mice reconstituted with Mac-CGI-58 Tg BM cells (ApoE^−/−/Tg-BM chimera) displayed a significant reduction of atherosclerosis lesions compared with control mice reconstituted with Mac-CGI-58 WT BM cells (ApoE^−/−/WT-BM chimera). Collectively, these data strongly suggest that CGI-58 overexpression in macrophages may protect against atherosclerosis development in mice.     

Chronic Ingestion of H1-Antihistamines Increase Progression of Atherosclerosis in Apolipoprotein E-/- Mice

Although increased serum histamine levels and H1R expression in the plaque are seen in atherosclerosis, it is not known whether H1R activation is a causative factor in the development of the disease, or is a host defense response to atherogenic signals. In order to elucidate how pharmacological inhibition of histamine receptor 1 (H1R) signaling affects atherogenesis, we administered either cetirizine (1 and 4 mg/kg. b.w) or fexofenadine (10 and 40 mg/kg. b.w) to ApoE^−/− mice maintained on a high fat diet for three months. Mice ingesting a low dose of cetirizine or fexofenadine had significantly higher plaque coverage in the aorta and cross-sectional lesion area at the aortic root. Surprisingly, the higher doses of cetirizine or fexofenadine did not enhance atherosclerotic lesion coverage over the controls. The low dose of fexofenadine, but not cetirizine, increased serum LDL cholesterol. Interestingly, the expression of iNOS and eNOS mRNA was increased in aortas of mice on high doses of cetirizine or fexofenadine. This may be a compensatory nitric oxide (NO)-mediated vasodilatory mechanism that accounts for the lack of increase in the progression of atherosclerosis. Although the administration of cetirizine did not alter blood pressure between the groups, there was a positive correlation between blood pressure and lesion/media ratio at the aortic root in mice receiving the low dose of cetirizine. However, this association was not observed in mice treated with the high dose of cetirizine or either doses of fexofenadine. The macrophages or T lymphocytes densities were not altered by low doses of H1-antihistamines, whereas, high doses decreased the number of macrophages but not T lymphocytes. The number of mast cells was decreased only in mice treated with low dose of fexofenadine. These results demonstrate that chronic ingestion of low therapeutic doses of cetirizine or fexofenadine enhance progression of atherosclerosis.     

Voluntary Exercise Stabilizes Established Angiotensin II-Dependent Atherosclerosis in Mice through Systemic Anti-Inflammatory Effects

We have previously demonstrated that exercise training prevents the development of Angiotensin (Ang) II-induced atherosclerosis and vulnerable plaques in Apolipoprotein E-deficient (ApoE^-/-) mice. In this report, we investigated whether exercise attenuates progression and promotes stability in pre-established vulnerable lesions. To this end, ApoE^-/- mice with already established Ang II-mediated advanced and vulnerable lesions (2-kidney, 1-clip [2K1C] renovascular hypertension model), were subjected to sedentary (SED) or voluntary wheel running training (EXE) regimens for 4 weeks. Mean blood pressure and plasma renin activity did not significantly differ between the two groups, while total plasma cholesterol significantly decreased in 2K1C EXE mice. Aortic plaque size was significantly reduced by 63% in 2K1C EXE compared to SED mice. Plaque stability score was significantly higher in 2K1C EXE mice than in SED ones. Aortic ICAM-1 mRNA expression was significantly down-regulated following EXE. Moreover, EXE significantly down-regulated splenic pro-inflammatory cytokines IL-18, and IL-1β mRNA expression while increasing that of anti-inflammatory cytokine IL-4. Reduction in plasma IL-18 levels was also observed in response to EXE. There was no significant difference in aortic and splenic Th1/Th2 and M1/M2 polarization markers mRNA expression between the two groups. Our results indicate that voluntary EXE is effective in slowing progression and promoting stabilization of pre-existing Ang II-dependent vulnerable lesions by ameliorating systemic inflammatory state. Our findings support a therapeutic role for voluntary EXE in patients with established atherosclerosis.     

Impact of Glutathione Peroxidase-1 Deficiency on Macrophage Foam Cell Formation and Proliferation: Implications for Atherogenesis

Clinical and experimental evidence suggests a protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx-1) in the atherogenic process. GPx-1 deficiency accelerates atherosclerosis and increases lesion cellularity in ApoE^−/− mice. However, the distribution of GPx-1 within the atherosclerotic lesion as well as the mechanisms leading to increased macrophage numbers in lesions is still unknown. Accordingly, the aims of the present study were (1) to analyze which cells express GPx-1 within atherosclerotic lesions and (2) to determine whether a lack of GPx-1 affects macrophage foam cell formation and cellular proliferation. Both in situ-hybridization and immunohistochemistry of lesions of the aortic sinus of ApoE^−/− mice after 12 weeks on a Western type diet revealed that both macrophages and – even though to a less extent – smooth muscle cells contribute to GPx-1 expression within atherosclerotic lesions. In isolated mouse peritoneal macrophages differentiated for 3 days with macrophage-colony-stimulating factor (MCSF), GPx-1 deficiency increased oxidized low density-lipoprotein (oxLDL) induced foam cell formation and led to increased proliferative activity of peritoneal macrophages. The MCSF- and oxLDL-induced proliferation of peritoneal macrophages from GPx-1^−/−ApoE^−/− mice was mediated by the p44/42 MAPK (p44/42 mitogen-activated protein kinase), namely ERK1/2 (extracellular-signal regulated kinase 1/2), signaling pathway as demonstrated by ERK1/2 signaling pathways inhibitors, Western blots on cell lysates with primary antibodies against total and phosphorylated ERK1/2, MEK1/2 (mitogen-activated protein kinase kinase 1/2), p90RSK (p90 ribosomal s6 kinase), p38 MAPK and SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase), and immunohistochemistry of mice atherosclerotic lesions with antibodies against phosphorylated ERK1/2, MEK1/2 and p90RSK. Representative effects of GPx-1 deficiency on both macrophage proliferation and MAPK phosphorylation could be abolished by the GPx mimic ebselen. The present study demonstrates that GPx-1 deficiency has a significant impact on macrophage foam cell formation and proliferation via the p44/42 MAPK (ERK1/2) pathway encouraging further studies on new therapeutic strategies against atherosclerosis.     

Overexpression of TGF-ß1 in macrophages reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice

Although macrophages represent the hallmark of both human and murine atherosclerotic lesions and have been shown to express TGF-ß1 (transforming growth factor β1) and its receptors, it has so far not been experimentally addressed whether the pleiotropic cytokine TGF-ß1 may influence atherogenesis by a macrophage specific mechanism. We developed transgenic mice with macrophage specific TGF-ß1 overexpression, crossed the transgenics to the atherosclerotic ApoE (apolipoprotein E) knock-out strain and quantitatively analyzed both atherosclerotic lesion development and composition of the resulting double mutants. Compared with control ApoE^−/− mice, animals with macrophage specific TGF-ß1 overexpression developed significantly less atherosclerosis after 24 weeks on the WTD (Western type diet) as indicated by aortic plaque area en face (p<0.05). Reduced atherosclerotic lesion development was associated with significantly less macrophages (p<0.05 after both 8 and 24 weeks on the WTD), significantly more smooth muscle cells (SMCs; p<0.01 after 24 weeks on the WTD), significantly more collagen (p<0.01 and p<0.05 after 16 and 24 weeks on the WTD, respectively) without significant differences of inner aortic arch intima thickness or the number of total macrophages in the mice pointing to a plaque stabilizing effect of macrophage-specific TGF-ß1 overexpression. Our data shows that macrophage specific TGF-ß1 overexpression reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice.     

Genetic deletion or TWEAK blocking antibody administration reduce atherosclerosis and enhance plaque stability in mice

Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. Tumour necrosis factor ligand superfamily member 12 (TNFSF12) also known as TNF-related weak inducer of apoptosis (TWEAK) is a proinflammatory cytokine that participates in atherosclerotic plaque development, but its role in plaque stability remains unclear. Using two different approaches, genetic deletion of TNFSF12 and treatment with a TWEAK blocking mAb in atherosclerosis-prone mice, we have analysed the effect of TWEAK inhibition on atherosclerotic plaques progression and stability. Mice lacking both TNFSF12 and Apolipoprotein E (TNFSF12^−/−ApoE^−/−) exhibited a diminished atherosclerotic burden and lesion size in their aorta. Advanced atherosclerotic plaques of TNFSF12^−/−ApoE^−/− or anti-TWEAK treated mice exhibited an increase collagen/lipid and vascular smooth muscle cell/macrophage ratios compared with TNFSF12^+/+ApoE^−/− control mice, reflecting a more stable plaque phenotype. These changes are related with two different mechanisms, reduction of the inflammatory response (chemokines expression and secretion and nuclear factor kappa B activation) and decrease of metalloproteinase activity in atherosclerotic plaques of TNFSF12^−/−ApoE^−/−. A similar phenotype was observed with anti-TWEAK mAb treatment in TNFSF12^+/+ApoE^−/− mice. Brachiocephalic arteries were also examined since they exhibit additional features akin to human atherosclerotic plaques associated with instability and rupture. Features of greater plaque stability including augmented collagen/lipid ratio, reduced macrophage content, and less presence of lateral xanthomas, buried caps, medial erosion, intraplaque haemorrhage and calcium content were present in TNFSF12^−/−ApoE^−/− or anti-TWEAK treatment in TNFSF12^+/+ApoE^−/− mice. Overall, our data indicate that anti-TWEAK treatment has the capacity to diminish proinflamatory response associated with atherosclerotic plaque progression and to alter plaque morphology towards a stable phenotype.     

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.     

Bromodomain-containing protein 7 deficiency augments atherosclerotic lesions in ApoE−/− mice

Atherosclerotic plaque formation is characterized by the persistence of lipid-laden macrophages on the inner walls of arteries. Chronic inflammation and imbalanced macrophage function are likely to play a critical role. Herein, we investigated whether bromodomain-containing protein 7 (Brd7), a member of the bromodomain-containing protein family, regulates atherosclerosis, and if so, which mechanisms are responsible for the process. We found that Brd7 is expressed in mouse atherosclerotic plaques, and mostly in macrophages. Inhibition of Brd7 accelerates atherosclerotic lesion formation in ApoE^?/? mice by promoting NF-κB–mediated inflammation. Furthermore, Brd7 inhibition alters the phenotype of macrophages and promotes plaque instability, at least partly via STAT6 signaling. Our data define a previously undescribed role of Brd7 in the development of atherosclerosis.