apoE-deficient Mice Research Articles

Heart function assessment during aging in apolipoprotein E knock-out mice

BACKGROUND: Apolipoprotein (apo) E isoforms have strong correlations with metabolic and cardiovascular diseases. However, it is not clear if apoE has a role in development of non-ischemic cardiomyopathy. Our study aims to analyze the involvement of apoE in non-ischemic cardiomyopathy. METHODS AND RESULTS: Serial echo-cardiographic measurements were performed in old wildtype and apoE deficient (apoE-/-) mice. Morphological and functional cardiac parameters were in normal range in both groups at the age of 12 month. At the age of 18 months, both groups had shown ventricular dilation and increased heart rates. However, the apoE-/- mice presented signs of diastolic dysfunction by hypertrophic changes in left ventricle, due probably to arterial hypertension. The right ventricle was not affected by age or genotype. CONCLUSION: Even in the absence of high fat diet, apoE deficiency in mice induces mild changes in the cardiac function of the left ventricle during aging, by developing diastolic dysfunction, which leads to heart failure with preserved ejection fraction. However, further studies are necessary to conclude over the role of apoE in cardiac physiology and its involvement in development of heart failure.

Potential of Gold Nanoparticles for Noninvasive Imaging and Therapy for Vascular Inflammation

PurposeMacrophages contribute to the progression of vascular inflammation, making them useful targets for imaging and treatment of vascular diseases. Gold nanoparticles (GNPs) are useful as computed tomography (CT) contrast agents and light absorbers in photothermal therapy. In this study, we aimed to assess the viability of macrophages incubated with GNPs after near-infrared (NIR) laser light exposure and to evaluate the utility of intravenously injected GNPs for in vivo imaging of vascular inflammation in mice using micro-CT.ProceduresMouse macrophage cells (RAW 264.7) were incubated with GNPs and assessed for GNP cellular uptake and cell viability before and after exposure to NIR laser light. For in vivo imaging, macrophage-rich atherosclerotic lesions were induced by carotid ligation in hyperlipidemic and diabetic FVB mice (n = 9). Abdominal aortic aneurysms (AAAs) were created by angiotensin II infusion in ApoE-deficient mice (n = 9). These mice were scanned with a micro-CT imaging system before and after the intravenous injection of GNPs.ResultsThe CT attenuation values of macrophages incubated with GNPs were significantly higher than those of cells incubated without GNPs (p < 0.04). Macrophages incubated with and without GNPs showed similar viability. The viability of macrophages incubated with GNPs (100 μg/ml or 200 μg/ml) was decreased by high-intensity NIR laser exposure but not by low-intensity NIR laser exposure. In vivo CT images showed higher CT attenuation values in diseased carotid arteries than in non-diseased contralateral arteries, although the difference was not statistically significant. The CT attenuation values of the perivascular area in AAAs of mice injected with GNPs were significantly higher than those of mice without injection (p = 0.0001).ConclusionsMacrophages with GNPs had reduced viability upon NIR laser exposure. GNPs intravenously injected into mice accumulated in sites of vascular inflammation, allowing detection of carotid atherosclerosis and AAAs in CT imaging. Thus, GNPs have potential as multifunctional biologically compatible particles for the detection and therapy of vascular inflammation.

P-selectin targeted RAGE-shRNA lipoplexes alleviate atherosclerosis-associated inflammation

The receptor for advanced glycation end products (RAGE) plays a central role in the chronic inflammatory process associated with atherosclerosis development. We aimed to develop lipoplexes carrying RAGE-short hairpin (sh) RNA, targeted to the adhesion molecule P-selectin, selectively expressed on the surface of activated endothelium (Psel-lipo/shRAGE) to down-regulate RAGE expression as a therapeutic strategy for atherosclerosis. In vitro, Psel-lipo/shRAGE lipoplexes were efficiently taken up by activated endothelial cells (EC), decreased the expression of RAGE protein, and proved to be functional by reducing the monocyte adhesion to activated EC. In ApoE-deficient mice, the targeted lipoplexes accumulated specifically and efficiently transfected the aorta. The repeated administration of Psel-lipo/shRAGE lipoplexes, twice per week for one month: i) reduced the expression of RAGE protein in the aorta by decreasing the expression of NF-kB and TNF-α; ii) diminished the plasma levels of TNF-α, IL6, IL-1β, and MCP-1; iii) inhibited the atherosclerotic plaque development and iv) had no significant adverse effects. In conclusion, the newly developed Psel-lipo/shRAGE lipoplexes reduce the inflammatory processes associated with RAGE signaling and the progression of atherosclerosis in ApoE-deficient mice. Downregulation of RAGE employing these lipoplexes may represent a promising new targeted therapy to block atherosclerosis progression.

Association of Pericyte Loss With Microthrombosis After Subarachnoid Hemorrhage in ApoE-Deficient Mice.

Background: The occurrence of microthrombosis contributes to not only delayed cerebral ischemia (DCI), but also early brain injury (EBI) after SAH. However, the underlying mechanism is not completely investigated. In the current study, we explored the underlying mechanism of microthrombosis in EBI stage after SAH in ApoE-deficient mice.Methods: Experimental SAH was established by endovascular perforation in apolipoprotein E (ApoE)-deficient mice and wild type (WT) mice. Neurobehavioral, molecular biological and histopathological methods were used to assess the relationship between pericytes loss, neurobehavioral performance, and microthrombosis.Results: We found that the number of microthrombi was significantly increased and peaked 48 h after SAH in WT mice. The increased microthrombosis was related to the decreased effective microcirculation perfusion area and EBI severity. ApoE-deficient mice showed more extensive microthrombosis than that of WT mice 48 h after SAH, which was thereby associated with greater neurobehavioral deficits. Immunohistochemical staining showed that microthrombi were predominantly located in microvessels where pericytes coverage was absent. Mechanistically, ApoE deficiency caused more extensive CypA-NF-κB-MMP-9 pathway activation than that observed in WT mice, which thereby led to more degradation of N-cadherin, and subsequently more pericytes loss. Thereafter, the major adhesion molecule that promoting microthrombi formation in microvessels, P-selectin, was considerably increased in WT mice and increased to a greater extent in the ApoE-deficient mice.Conclusion: Taken together, these data suggest that pericytes loss is associated with EBI after SAH through promoting microthrombosis. Therapies that target ApoE to reduce microthrombosis may be a promising strategy for SAH treatment.

Abstract P101: Suppression Of Serum Amyloid A Limits Progression Of Obesity Associated Abdominal Aortic Aneurysms

Obesity increases the risk for abdominal aortic aneurysms (AAA) in humans, and enhances angiotensin II (AngII)-induced AAA formation in C57BL/6 mice. Obesity is also associated with increases in serum amyloid A (SAA). We previously reported that deficiency of SAA significantly reduces AngII-induced inflammation and AAA in apoE-deficient mice. In this study we investigated whether SAA plays a role in progression of an established AAA in obese C57BL/6 mice. Approach and Results: Male C57BL/6 mice were fed a high fat diet (60% kcal as fat) throughout the study. After 4 months of diet the mice were infused with angiotensin II (AngII) at 1000ng/kg/min until the end of the study. Ultrasound (US) was performed in all mice before and after 28 days of AngII infusion, and mice that had at least a 25% increase in the luminal diameter of the abdominal aorta were stratified by luminal diameter into 3 groups. Group 1 was killed to establish baseline AAA. Groups 2 and 3 continued to receive AngII for a further 8 weeks along with an antisense oligonucleotide (ASO) that suppresses all 3 acute phase SAA isoforms (SAA-ASO), or a control ASO (5 mg/kg/wk). US was repeated at study end to assess AAA progression. Plasma SAA at the end of the experiment was 89.2±83.2 mg/L in the control ASO group, and 18.6±0.7 mg/L in the SAA-ASO group (mean±SD , p=0.008). There was no impact of SAA suppression on body weight, body fat, or blood pressure. After the first 4 weeks of AngII infusion, the average luminal diameter in all mice was 1.81±0.40 mm (mean±SD). Mice that received the control ASO had continued aortic dilation (average luminal aortic diameter 2.06±0.42 mm), whereas the mice that received the SAA-ASO had a significant reduction in progression of aortic dilation (average luminal diameter 1.64±0.43 mm, p=0.0015 for interaction between time and group). Conclusions: We demonstrate for the first time that suppression of SAA protects obese C57BL/6 mice from progression of AngII-induced AAA. Suppression of SAA may be a therapeutic approach to limit AAA progression.

Abstract MP42: Macrophage Chromatin Remodeling Complex Subunit Baf60a In Atherosclerosis

Objectives: Atherosclerosis is a primary medical concern due to the increasing prevalence and lack of effective treatment that reverses the disease progression. While multiple genetic mutations/variations have been associated with atherosclerotic risk, the development of atherosclerosis in response to risk factors is also contributed by factors at the epigenetic and chromatin level. The SWI/SNF chromatin remodeling complex alters DNA accessibility and chromatin structure to modulate gene expression in response to external and internal cellular environments, potentially serving as an underexplored compartment of CVD and atherosclerosis development. Approach and Results: In an effort to identify key chromatin remodeling compartments involved in atherosclerotic foam cell formation, primary bone marrow-derived macrophages (BMDMs) were exposed to a pro-atherogenic environment, specifically to oxidized Low-density lipoprotein (oxLDL). We observed drastically and time-dependent decrease in the protein abundance of BAF60a, a transcription factor interacting subunit of the SWI/SNF chromatin remodeling complex. Macrophage-specific Baf60a deletion in ApoE deficient mice showed over 30% increase in aortic plaque formation after 16-weeks of western diet feeding (N=12, p=0.0097). Integrated analysis of RNA-seq and ATAC-seq regulatory network in oxLDL treated BAF60a deficient BMDMs identified a shift of chromatin accessibility, transcription factor footprint, and a reshape in gene expression patterns that regulate extracellular matrix organization, focal adhesion, integrin signaling, inflammation, and lead to foam cell formation which ultimately contribute to the atherosclerotic plaque build-up. Conclusions: Our data demonstrated the role of macrophage BAF60a in atherogenesis and suggested a potential therapeutic target to treat atherosclerosis, presumably by compensating for the loss of Baf60a.

Saringosterol from Sargassum fusiforme Modulates Cholesterol Metabolism and Alleviates Atherosclerosis in ApoE-Deficient Mice.

Dysregulation of cholesterol homeostasis is a major risk factor of atherosclerosis, which can lead to serious health problems, including heart attack and stroke. Liver X receptor (LXR) α and β are transcription factors belonging to the nuclear receptor superfamily, which play important roles in cholesterol homeostasis. Selectively activating LXRβ provides a promising strategy for the treatment of atherosclerosis. Here, we employed atherosclerotic apoE-knockout mice to evaluate the effects of saringosterol, a phytosterol with potent and selective action for LXRβ, which we identified previously in edible marine seaweed Sargassum fusiforme. We found that saringosterol treatment reduced the atherosclerotic plaque burden without having undesirable adverse hepatic effects in apoE-deficient mice fed an atherogenic diet. Meanwhile, reduced serum levels of cholesterol, accompanied by altered expression of LXR-regulated genes involved in cholesterol absorption, transport, efflux, excretion, and elimination, were observed in apoE-knockout mice after saringosterol treatment. Together, our study not only establishes saringosterol as an effective cholesterol-lowering and anti-atherogenic phytosterol but also provides insights into the underlying mechanism.

Modulation of Peripheral CD4+CD25+Foxp3+ Regulatory T Cells Ameliorates Surgical Stress-Induced Atherosclerotic Plaque Progression in ApoE-Deficient Mice.

Systemic inflammation associated with major surgery rapidly accelerates atherosclerotic plaque progression in mice. Regulatory T cells (Tregs) have emerged as important modulators of atherogenesis. In coronary artery disease patients, low frequency of Tregs constitutes an independent risk factor for cardiovascular complications after non-cardiac surgery. In this exploratory analysis, we investigate whether preoperative Treg levels affect surgery-induced atherosclerotic lesion destabilization in a murine model of perioperative stress. After 9 weeks of high-cholesterol diet, atherosclerotic apolipoprotein E-deficient mice with modulated Treg levels were subjected to a 30-minute surgical procedure consisting of general isoflurane anesthesia, laparotomy and moderate blood loss. Controls underwent general anesthesia only. Brachiocephalic arteries were harvested 3 days after the intervention for histomorphological analyses of atherosclerotic plaques. Tregs were depleted by a single dose of anti-CD25 monoclonal antibody (mAb) administered 6 days prior to the intervention. Expansion of Tregs was induced by daily injections of IL-2/anti-IL-2 complex (IL-2C) on three consecutive days starting 3 days before surgery. Isotype-matched antibodies and PBS served as controls. Antibody-mediated modulation was Treg-specific. IL-2C treatment resulted in an eight-fold elevation of peripheral CD4+CD25+Foxp3+ Tregs compared to mice administered with anti-CD25 mAb. In mice treated with PBS and anti-CD25 mAb, surgical stress response caused a significant increase of atherosclerotic plaque necrosis (PBS: p < 0.001; anti-CD25 mAb: p = 0.037). Preoperative Treg expansion abrogated perioperative necrotic core formation (p = 0.556) and significantly enhanced postoperative atherosclerotic plaque stability compared to PBS-treated mice (p = 0.036). Postoperative plaque volume (p = 0.960), stenosis (p = 0.693), lesional collagen (p = 0.258), as well as the relative macrophage (p = 0.625) and smooth muscle cell content (p = 0.178) remained largely unaffected by preoperative Treg levels. In atherosclerotic mice, therapeutic expansion of Tregs prior to major surgery mitigates rapid effects on perioperative stress-driven atherosclerotic plaque destabilization. Future studies will show, whether short-term interventions modulating perioperative inflammation qualify for prevention of cardiovascular events associated with major non-cardiac surgery.

The extract of Ilex kudingcha inhibits atherosclerosis in apoE-deficient mice by suppressing cholesterol accumulation in macrophages.

It was previously reported that oleanolic acid and ursolic acid, triterpenoid compounds occurring in Ilex kudingcha, ameliorate hyperlipidemia and atherosclerosis in apoE-deficient mice. In the present study, we investigated whether I. kudingcha extract exerts similar inhibitory effects on cholesterol accumulation in human monocyte-derived macrophages (HMDMs) and atherogenesis in apoE-deficient mice. I. kudingcha extract significantly inhibited cholesterol ester (CE) accumulation induced by acetylated LDL (acetyl-LDL) in HMDMs; however, it generated no effect on cell viability in HMDMs. I. kudingcha extract also suppressed CE accumulation in acyl-CoA:cholesterol acyl-transferase (ACAT)-overexpressing Chinese hamster ovary (CHO) cells, thereby indicating that it inhibits ACAT activity. Furthermore, the oral administration of I. kudingcha extract to apoE-deficient mice significantly decreased the levels of serum cholesterol, triglyceride, sLOX-1, as well as the regions of atherosclerotic lesions in the mice. Our study reveals crucial new-found evidence that I. kudingcha extract significantly inhibits ACAT activity and suppresses atherogenesis.

LncRNA Miat knockdown alleviates endothelial cell injury through regulation of miR-214-3p/Caspase-1 signalling during atherogenesis.

Atherosclerosis is a common problem in healthy people around the world. Long noncoding RNAs (lncRNAs) play important roles in atherosclerosis. Myocardial infarction-associated transcript (Miat) is a cardiovascular disease-associated lncRNA. Its role and mechanism in atherosclerosis is still not fully clarified. Our study aims to explore the role and mechanism of lncRNA Miat in atherosclerosis. The atherosclerosis models were established both in vitro and in vivo. Real-time PCR was used to measure the expression of lncRNA Miat, miR-214, Caspase-1 and IL-1β. Western blot was performed to detect the protein expression of Caspase-1. CCK-8 assay, Tunel staining, and flow cytometry analysis were conducted to detect proliferation and apoptosis of human aortic endothelial cells (HAECs), respectively. Oil red O staining and HE staining were used to evaluated the histological changes of the aorta. The results found that lncRNA Miat was upregulated in ox-LDL-induced atherosclerosis model in vitro. The inhibition of lncRNA Miat protects against ox-LDL-induced HAEC injury, presented as increased cell viability and decreased apoptosis. LncRNA Miat and miR-214has binding site, and CASP1, which encodes Caspase-1, is a target of miR-214. The downregulation of lncRNA Miat increased the expression of miR-214-3p and decreased the expression of Caspase-1, as well as its downstream molecule IL-1β in HAECs. However, the inhibition of miR-214-3p attenuated the effect of lncRNA Miat downregulation on HAECs. Furthermore, the downregulation of lncRNA Miat alleviated atherosclerosis in ApoE-deficient mice. Correspondingly, the expression of miR-214-3p was upregulated and Caspase-1 was downregulated after knockdown of lncRNA Miat. In conclusion, downregulation of lncRNA Miat exerts a protective effect against atherosclerosis through the regulation miR-214-3p/Caspase-1signalling pathway. Therefore, the inhibition of lncRNA Miat expression may be an effective strategy in the treatment of atherosclerosis.

The angiopoietin receptor Tie2 in arterial endothelium inhibits atherosclerosis

Background and Aims: Leukocytes and resident cells in the arterial wall participate in the buildup of atherosclerotic plaques in areas of turbulent blood flow. Deletion of the endothelial Tie1 receptor, which acts in a complex with Tie2 as the receptor for angiopoietins, was reported to reduce the atherosclerotic burden and decrease inflammation in aortas in ApoE deficient mice. In this study, we aimed to evaluate the function of the Tie2 gene in atherosclerosis.

Apolipoprotein E deficiency activates thermogenesis of white adipose tissues in mice through enhancing β-hydroxybutyrate production from precursor cells.

White adipose tissue (WAT) has the capacity to undergo a white-to-beige phenotypic switch, known as browning, in response to stimuli such as cold. However, the mechanism underlying beige adipocyte formation is largely unknown. Apolipoprotein E (ApoE) is highly induced in WAT and has been implicated in lipid metabolism. Here, we show that ApoE deficiency in mice increased oxygen consumption and thermogenesis and enhanced adipose browning pattern in inguinal WAT (iWAT), with associated characteristics such as increased Ucp1 and Pparγ expression. At the cellular level, ApoE deficient beige adipocytes had increased glucose uptake and higher mitochondrial respiration than wild-type cells. Mechanistically, we showed that ApoE deficient iWAT and primary adipose precursor cells activated the thermogenic genes program by stimulating the production of ketone body β-hydroxybutyrate (βHB), a novel adipose browning promoting factor. This was accompanied by increased expression of genes involved in ketogenesis and could be compromised by the treatment for ketogenesis inhibitors. Consistently, ApoE deficient mice show higher serum βHB level than wild-type mice in the fed state and during cold exposure. Our results further demonstrate that the increased βHB production in ApoE deficient adipose precursor cells could be attributed, at least in part, to enhanced Cd36 expression and CD36-mediated fatty acid utilization. Our findings uncover a previously uncharacterized role for ApoE in energy homeostasis via its cell-autonomous action in WAT.

Overexpressing low-density lipoprotein receptor reduces tau-associated neurodegeneration in relation to apoE-linked mechanisms

APOE is the strongest genetic risk factor for late-onset Alzheimer's disease. ApoE exacerbates tau-associated neurodegeneration by driving microglial activation. However, how apoE regulates microglial activation and whether targeting apoE is therapeutically beneficial in tauopathy is unclear. Here, we show that overexpressing an apoE metabolic receptor, LDLR (low-density lipoprotein receptor), in P301S tauopathy mice markedly reduces brain apoE and ameliorates tau pathology and neurodegeneration. LDLR overexpression (OX) in microglia cell-autonomously downregulates microglial Apoe expression and is associated with suppressed microglial activation as in apoE-deficient microglia. ApoE deficiency and LDLR OX strongly drive microglial immunometabolism toward enhanced catabolism over anabolism, whereas LDLR-overexpressing microglia also uniquely upregulate specific ion channels and neurotransmitter receptors upon activation. ApoE-deficient and LDLR-overexpressing mice harbor enlarged pools of oligodendrocyte progenitor cells (OPCs) and show greater preservation of myelin integrity under neurodegenerative conditions. They also show less reactive astrocyte activation in the setting of tauopathy.

Procyanidin B2 Reduces Vascular Calcification through Inactivation of ERK1/2-RUNX2 Pathway.

Vascular calcification is strongly associated with atherosclerotic plaque burden and plaque instability. The activation of extracellular signal-regulated kinase 1/2 (ERK1/2) increases runt related transcription factor 2 (RUNX2) expression to promote vascular calcification. Procyanidin B2 (PB2), a potent antioxidant, can inhibit ERK1/2 activation in human aortic smooth muscle cells (HASMCs). However, the effects and involved mechanisms of PB2 on atherosclerotic calcification remain unknown. In current study, we fed apoE-deficient (apoE−/−) mice a high-fat diet (HFD) while treating the animals with PB2 for 18 weeks. At the end of the study, we collected blood and aorta samples to determine atherosclerosis and vascular calcification. We found PB2 treatment decreased lesions in en face aorta, thoracic, and abdominal aortas by 21.4, 24.6, and 33.5%, respectively, and reduced sinus lesions in the aortic root by 17.1%. PB2 also increased α-smooth muscle actin expression and collagen content in lesion areas. In the aortic root, PB2 reduced atherosclerotic calcification areas by 75.8%. In vitro, PB2 inhibited inorganic phosphate-induced osteogenesis in HASMCs and aortic rings. Mechanistically, the expression of bone morphogenetic protein 2 and RUNX2 were markedly downregulated by PB2 treatment. Additionally, PB2 inhibited ERK1/2 phosphorylation in the aortic root plaques of apoE−/− mice and calcified HASMCs. Reciprocally, the activation of ERK1/2 phosphorylation by C2-MEK1-mut or epidermal growth factor can partially restore the PB2-inhibited RUNX2 expression or HASMC calcification. In conclusion, our study demonstrates that PB2 inhibits vascular calcification through the inactivation of the ERK1/2-RUNX2 pathway. Our study also suggests that PB2 can be a potential option for vascular calcification treatment.

Regular exercise stimulates endothelium autophagy via IL-1 signaling in ApoE deficient mice.

Regular exercise maintains arterial endothelial cell homeostasis and protects the arteries from vascular disease, such as peripheral artery disease and atherosclerosis. Autophagy, which is a cellular process that degrades misfolded or aggregate proteins and damaged organelles, plays an important role in maintaining organ and cellular homeostasis. However, it is unknown whether regular exercise stimulates autophagy in aorta endothelial cells of mice prone to atherosclerosis independently of their circulating lipid profile. Here, we observed that 16weeks of voluntary exercise reduced high-fat diet-induced atherosclerotic plaque formation in the aortic root of ApoE deficient mice, and that this protection occurred without changes in circulating triglycerides, total cholesterol, and lipoproteins. Immunofluorescence analysis indicated that voluntary exercise increased levels of the autophagy protein LC3 in aortic endothelial cells. Interestingly, human umbilical vein endothelial cells (HUVECs) exposed to serum from voluntarily exercised mice displayed significantly increased LC3-I and LC3-II protein levels. Analysis of circulating cytokines demonstrated that voluntary exercise caused changes directly relevant to IL-1 signaling (ie, decreased interleukin-1 receptor antagonist [IL-1ra] while also increasing IL-1α). HUVECs exposed to IL-1α and IL-1β recombinant protein significantly increased LC3 mRNA expression, LC3-I and LC3-II protein levels, and autophagy flux. Collectively, these results suggest that regular exercise protects arteries from ApoE deficient mice against atherosclerosis at least in part by stimulating endothelial cell autophagy via enhanced IL-1 signaling.

Dietary Avian Proteins Are Comparable to Soybean Proteins on the Atherosclerosis Development and Fatty Liver Disease in Apoe-Deficient Mice.

Background and aim: The type and amount of dietary protein has become a topic of renewed interest in light of their involvement in metabolic diseases, atherosclerosis and thrombosis. However, little attention has been devoted to the effect of avian proteins despite their wide human consumption. The aim was to investigate the influence of chicken and turkey as sources of protein compared with that of soybean on atherosclerosis and fatty liver disease. Methods and results: To this purpose, male and female Apoe-deficient were fed purified Western diets differing in their protein sources for 12 weeks. After this period, blood, liver, aortic tree and heart base samples were taken for analyses of plasma lipids and atherosclerosis. Plasma triglycerides, non-esterified fatty acids, esterified cholesterol levels and radical oxygen species in lipoproteins changed depending on the diet and sex. Females consuming the turkey protein-containing diet showed decreased atherosclerotic foci, as evidenced by the en face atherosclerosis analyses. The presence of macrophages and smooth muscle cells in plaques were not modified, and no changes were observed in hepatic lipid droplets in the studied groups either. Paraoxonase activity was higher in the group consuming turkey protein without sex differences, but only in females, it was significantly associated with aortic lesion areas. Conclusions: Compared to soybean protein, the consumption of avian proteins depending on sex resulted in similar or lower atherosclerosis development and comparable hepatic steatosis.

G protein-coupled estrogen receptor GPR30 exerts vasoprotective effects in apolipoprotein E-deficient mice

IntroductionGPR30 is an intracellular transmembrane G protein-coupled receptor that mediates non-genomic estrogen signaling. The GPR30 agonist G-1 modulates glucose homeostasis and vascular function. However, its impact on vascular inflammation and atherogenesis has not yet been investigated in the atherosclerotic apolipoprotein E-deficient(ApoE-/-) mouse model.Material and methodsApoE-/- mice were fed a high-cholesterol diet for 7 weeks while being treated with the selective GPR30 agonist G-1 (n=6-7). After the treatment period, vascular relaxation capacity, vascular oxidative stress, and atherosclerotic plaque burden were assessed. In vitro, reactive oxygen species, expression levels of the angiotensin II type1(AT1) receptor, and proliferation rate were quantified in human coronary artery smooth muscle cells(HCASMC).ResultsG-1 significantly improved glucose tolerance in vivo (142.2±8.1mg/dl vs. 204.6±13.3mg/dl), G-1 reduced vascular oxidative stress (221±88RLU/s/mg vs.1,983±885RLU/s/mg) and improved endothelium-dependent vasodilation (relaxation to 35.1±4.5% vs.63.0±4.6%). Furthermore, treatment with G-1 significantly reduced the atherosclerotic plaque burden of female ApoE-/- mice (56.5±3.7% vs.75.5±2.9%). In vitro, G-1 provoked a significant downregulation of the AT1 receptor in HCASMC (0.67±0.09-fold). Furthermore, G-1 blunted angiotensin II-induced ROS production by HCASMC (817±7RLU/s/mg vs.1,625±105 RLU/s/mg) and diminished HCASMC proliferation (-26.8±2.7% vs.+50.4±1.7%).ConclusionsSelective GPR30 activation improves glucose tolerance in vivo and decreases vascular ROS production in vitro and in vivo. In vitro, the antioxidant effect might be mediated by downregulation of the AT1 receptor. In vivo, the antioxidant effect of G-1 is associated with an improved endothelial function and a reduced atherosclerotic plaque burden in ApoE-deficient mice, indicating beneficial vascular effects of GPR30 activation. GPR30 agonism might thus be a compelling treatment strategy against atherosclerosis.

Macrophage Specific IGF‐1 Overexpression Decreases Atherosclerosis, CXCL12 Chemokine, And Increases Cholesterol Efflux In ApoE Deficient Mice

Increased bioavailability of Insulin Like Growth Factor (IGF-1) has been shown to decrease cardiovascular event risk. We have shown that systemic IGF-1 administration reduced atherosclerosis in ApoE deficient (Apoe-/-) mice fed a high fat diet and that those animals have reduced plaque macrophages. Thus, we have shown that IGF-1 is atheroprotective, but cell-specific mechanism(s) mediating IGF-1's effect need to be identified. IGF-1 interacts with major cell types in atherosclerotic plaque: macrophages, smooth muscle cells, and endothelial cells. Results of our previous in vitro experiments suggest that macrophages play a predominant role in mediating IGF-1 effects. We hypothesized that increasing IGF-1 levels strictly in macrophages will prevent atherosclerosis. Using a novel macrophage-specific IGF-1 overexpressing transgenic mouse bred to an Apoe-/- background (MF-IGF1 mice), we assessed atherosclerotic plaque burden, stability, and monocyte recruitment. We isolated plaque using laser capture microdissection and we analyzed monocyte recruitment via intravenous injection of fluorescent red beads among other traditional techniques. Macrophage IGF-1 overexpression downregulated plaque burden by 30% (P<0.01), reduced plaque macrophages by 47% (P<0.001) and promoted a stable plaque phenotype. Monocyte recruitment was reduced by 70% (P<0.05) in MF-IGF1 mice and was associated with a decrease in circulating levels of CXC Chemokine Ligand 12 (CXCL12) (27% reduction compared to control, P<0.05). CXCL12 protein levels were reduced in plaque and peritoneal macrophages in MF-IGF1 mice. IGF-1 completely blocked oxidized low-density lipoprotein (oxLDL)-dependent increase of CXCL12 mRNA transcription (98% reduction vs. control, P<0.01) and IGF-1 treatment reduced CXCL12 protein (56% decrease vs. control, P<0.001)in vitro. ATP-binding cassette transporter A1 (ABCA1) is the key cholesterol transporter mediating macrophage cholesterol efflux. We found that peritoneal macrophages isolated from MF-IGF1 mice have a 2-fold increase in ABCA1 protein levels. To study whether IGF-1 changes macrophage cholesterol efflux, we loaded THP-1 cells with oxLDL and then attached a fluorescent label to measure changes in efflux. We found a 27% increase in cholesterol efflux in IGF-1 (100ng/mL)-treated cells (P<0.01) when using Apolipoprotein A I as a cholesterol acceptor. Overall, our results indicate that macrophage IGF-1 reduces atherosclerosis and decreases CXCL12, a chemokine newly implicated in atheroprogression. CXCL12 downregulates ABCA1 expression. IGF-1 potentially exerts its atheroprotective effect via this reduction in CXCL12 by reducing monocyte recruitment and by increasing ABCA1 and thereby increasing cholesterol efflux capacity.

The effect of cold exposure on serum cholesterol is dependent upon ApoE.

Several lines of evidence indicate that cold stimulation may not only activate brown adipose tissue (BAT) and the white adipose tissue (WAT), but also regulate the lipid metabolism and influence the development of atherosclerosis. However, the study of cold exposure affecting cholesterol metabolism have opposite results in different experiments, and Apolipoprotein E (ApoE) may play an important role. There is still a lack of complete research to illustrate this problem. In this study, we first analyzed and discussed the activation of interscapular brown adipose tissue (iBAT), inguinal white adipose tissue (iWAT) and epididymal white adipose tissue (eWAT) under cold exposure (4°C) in male wild-type C57BL/6J (WT) and ApoE-deficient mice (ApoE-/-) fed high-fat diet (HFD) for 4 weeks. Subsequently, we investigated the effect of cold exposure on blood lipid profiles in both models. We further explored whether cold exposure can reduce serum cholesterol. In both WT and ApoE-/- mice, cold exposure activates iBAT and iWAT, as well as hardly affects eWAT. In WT mice,4 weeks cold exposure (4°C) reduces serum triglyceride by 28%, cholesterol by 30% and LDL-cholesterol by 63%. In ApoE-/- mice, cold stimulation decreases serum triglyceride by 59%, but increases cholesterol by 20% and LDL-cholesterol by 25%. Based on these findings, we conclude that cold exposure decreases serum cholesterol is dependent upon the existence of ApoE.

Adipocyte-Specific ACKR3 Regulates Lipid Levels in Adipose Tissue.

Dysfunctional adipose tissue (AT) may contribute to the pathology of several metabolic diseases through altered lipid metabolism, insulin resistance, and inflammation. Atypical chemokine receptor 3 (ACKR3) expression was shown to increase in AT during obesity, and its ubiquitous elimination caused hyperlipidemia in mice. Although these findings point towards a role of ACKR3 in the regulation of lipid levels, the role of adipocyte-specific ACKR3 has not yet been studied exclusively in this context. In this study, we established adipocyte- and hepatocyte-specific knockouts of Ackr3 in ApoE-deficient mice in order to determine its impact on lipid levels under hyperlipidemic conditions. We show for the first time that adipocyte-specific deletion of Ackr3 results in reduced AT triglyceride and cholesterol content in ApoE-deficient mice, which coincides with increased peroxisome proliferator-activated receptor-γ (PPAR-γ) and increased Angptl4 expression. The role of adipocyte ACKR3 in lipid handling seems to be tissue-specific as hepatocyte ACKR3 deficiency did not demonstrate comparable effects. In summary, adipocyte-specific ACKR3 seems to regulate AT lipid levels in hyperlipidemic Apoe−/− mice, which may therefore be a significant determinant of AT health. Further studies are needed to explore the potential systemic or metabolic effects that adipocyte ACKR3 might have in associated disease models.