ApoE Knockout Mice Research Articles

Low-Dose Gallic Acid Administration Does Not Improve Diet-Induced Metabolic Disorders and Atherosclerosis in Apoe Knockout Mice.

Diets rich in polyphenols are known to be beneficial for cardiovascular health. Gallic acid (GA) is a plant-derived triphenolic chemical with multiple cardio-protective properties, such as antiobesity, anti-inflammation, and antioxidation. However, whether GA could protect against atherosclerotic cardiovascular diseases is still not defined. Here, we investigated the effects of low-dose GA administration on diet-induced metabolic disorders and atherosclerosis in the atherosclerosis-prone apolipoprotein E (Apoe) knockout mice fed on a high-fat Western-type diet (WTD) for 8 weeks. Our data showed that GA administration by oral gavage at a daily dosage of 20 mg/kg body weight did not significantly ameliorate WTD-induced hyperlipidemia, hepatosteatosis, adipogenesis, or insulin resistance; furthermore, GA administration did not significantly ameliorate WTD-induced atherosclerosis. In conclusion, our data demonstrate that low-dose GA administration does not elicit significant health effect on diet-induced metabolic disorders or atherosclerosis in the Apoe knockout mice. Whether GA could be beneficial for atherosclerotic cardiovascular diseases therefore needs further exploration.

Targeting Peptide, Fluorescent Reagent Modified Magnetic Liposomes Coated with Rapamycin Target Early Atherosclerotic Plaque and Therapy.

Atherosclerosis is the leading cause of global morbidity and mortality. Its therapy requires research in several areas, such as diagnosis of early arteriosclerosis, improvement of the pharmacokinetics and bioavailability of rapamycin as its therapeutic agents. Here, we used the targeting peptide VHPKQHR (VHP) (or fluorescent reagent) to modify the phospholipid molecules to target vascular cell adhesion molecule-1 (VCAM-1) and loaded ultrasmall paramagnetic iron oxide (USPIO/Fe3O4) plus rapamycin (Rap) to Rap/Fe3O4@VHP-Lipo (VHPKQHR-modified magnetic liposomes coated with Rap). This nanoparticle can be used for both the diagnosis and therapy of early atherosclerosis. We designed both an ex vivo system with mouse aortic endothelial cells (MAECs) and an in vivo system with ApoE knockout mice to test the labeling and delivering potential of Rap/Fe3O4@VHP-Lipo with fluorescent microscopy, flow cytometry and MRI. Our results of MRI imaging and fluorescence imaging showed that the T2 relaxation time of the Rap/Fe3O4@VHP-Lipo group was reduced by 2.7 times and 1.5 times, and the fluorescence intensity increased by 3.4 times and 2.5 times, respectively, compared with the normal saline group and the control liposome treatment group. It showed that Rap/Fe3O4@VHP-Lipo realized the diagnosis of early AS. Additionally, our results showed that, compared with the normal saline and control liposomes treatment group, the aortic fluorescence intensity of the Rap/Fe3O4@VHP-Lipo treatment group was significantly weaker, and the T2 relaxation time was prolonged by 8.9 times and 2.0 times, indicating that the targeted diagnostic agent detected the least plaques in the Rap/Fe3O4@VHP-Lipo treatment group. Based on our results, the synthesized theragnostic Rap/Fe3O4@VHP-Lipo serves as a great label for both MRI and fluorescence bimodal imaging of atherosclerosis. It also has therapeutic effects for the early treatment of atherosclerosis, and it has great potential for early diagnosis and can achieve the same level of therapy with a lower dose of Rap.

Abstract 231: Monocyte-mimicking Nanoparticles For Atherosclerosis-targeted Therapy

Atherosclerosis, characterized by plaque buildup in arteries, is a major cause of cardiovascular mortality globally. Despite advances in diagnostics and interventions over the past few decades, the treatment options and outcomes remain far less than optimal. Nanotechnology has demonstrated emerging success in clinical settings; however, a potent targeted nanotherapeutic for atherosclerosis remains underdeveloped. In this study, we designed a new class of nanocarriers mimicking circulating monocyte features to enhance the site-specific delivery of theranostic agents for atherosclerosis. We first synthesized polymeric cores encapsulating a fluorescent payload with a modified nanoprecipitation method and cloaked the polymeric cores (NPs) with the plasma membrane fraction isolated from mouse monocytes. Our characterization results verified that NP cores are covered with a uniform lipid layer and that the resulting monocyte-mimicking nanoparticles (MNPs) retain the membrane proteins on their surface and have a similar value of zeta potential as monocytes. Both MNPs and NPs did not exhibit any hemotoxicity in vitro ; however, when incubated with cultured human vascular endothelial cells (ECs), MNPs showed a significantly higher uptake efficiency by ECs than NPs. Moreover, our in vivo studies with ApoE-knockout mice indicates that MNPs accumulated only in the atherosclerotic arteries but no other areas of the vasculature when administered intravenously. To summarize, our findings strongly support that monocyte membrane cloaking facilitates the nanoparticle attachment to atherosclerotic regions and enhances the entry of nanoparticles into the inflammatory endothelium in the arteries, suggesting that MNPs would serve as an excellent delivery strategy for targeted atherosclerosis therapy.

Abstract 436: Loss Of Serine Hydroxymethyltransferase 2 In Macrophages Enhances Atherosclerosis While Reducing Macrophage Mitochondrial Respiration

Despite significant advancements in therapeutics and intervention, cardiovascular diseases remain the leading cause of death worldwide. Atherosclerosis, the accumulation of lipid-laden plaques in large arteries, is the underlying cause of most cardiovascular diseases. While the role of lipids is well-established in atherosclerotic cardiovascular disease, the contribution of amino acids is becoming increasingly recognized. Indeed, lower circulating glycine is consistently reported in patients with cardiovascular diseases. The mitochondrial serine hydroxymethyltransferase (SHMT2) is responsible for the bulk of glycine biosynthesis in most cell types. SHMT2 catalyzes the formation of glycine from serine and drives one-carbon metabolism necessary for mitochondrial protein translation. SHMT2 is critical in development since global knockout mice are nonviable. Previous studies showed patients with significant coronary artery disease exhibit a reduced glycine:serine ratio compared with healthy individuals. We discovered LPS-treated macrophages similarly attenuated glycine:serine concomitant with reduced SHMT2 expression. Moreover, we found SHMT2 is highly expressed in plaque-associated macrophages in both human and mouse atherosclerotic plaques. We hypothesized that SHMT2 regulates macrophage inflammatory phenotype in atherosclerosis through alterations in mitochondrial function. Deletion of SHMT2 reduced mitochondrial respiration while enhancing glycolysis similar to LPS-treated macrophages. In addition, SHMT2 deletion augmented oxidized-LDL accumulation in macrophages. To determine whether macrophage-expressed SHMT2 contributes to atherosclerosis, we applied CRISPR/Cas9 and Cre-lox recombination system to generate myeloid-specific SHMT2 knockout ( Shmt2 MKO ) mice. We further crossed Shmt2 MKO mice with Apoe knockout ( Apoe -/- ) mice to study atherosclerosis following Western diet feeding. Compared to littermate controls ( Shmt2 fl / Apoe -/- ), Shmt2 MKO / Apoe -/- mice had significantly enhanced plaque area. Our findings, representing the first investigation of conditional SHMT2 knockout mice, suggest a novel immunometabolic role for SHMT2 in macrophages in atherosclerosis.

Enhancement of Endothelial KCa Channel Activity as a Novel Strategy to Oppose Atherosclerosis

Atherosclerosis represents a major risk factor for cardiovascular disease and is associated with endothelial dysfunction (ED), which facilitates fatty plaque formation, impairs blood flow and increases arterial stiffness. A hallmark feature of ED is decreased bioavailability of nitric oxide (NO). We have previously reported that pharmacological activation of endothelial Ca2+‐activated K+ channels (KCa2.3 and KCa3.1) can oppose ED by enhancing endothelium‐dependent vasodilation.We hypothesized that improving endothelial function will mitigate the development and/or severity of atherosclerosis in Apoe knockout (Apoe‐/‐) mice. Administration of the KCa channel activator SKA‐31 was utilized to improve endothelial function in vivo.Experimentally, male, 8‐week old Apoe‐/‐ mice on a regular chow diet were administered one of three daily treatments for 16 weeks: SKA‐31 (10 mg/kg), the KCa3.1 channel blocker senicapoc (40 mg/kg), or drug vehicle alone. Pharmacological inhibition of KCa3.1 channels is reported to reduce atherosclerosis in Apoe‐/‐ mice and was utilized as a benchmark. Drugs were formulated in miglyol and condensed milk, which was readily ingested by the mice. Cardiac function and arterial pulse wave velocity (PWV) were assessed by echocardiography under isoflurane anesthesia. Atherosclerotic lesions in the aorta were visualized by Oil‐Red‐O staining and select tissue histology was carried out using H&E staining. Abdominal aortic contractility and relaxation were measured by wire myography in drug‐treated Apoe‐/‐ mice and age/sex‐matched wild‐type C57/BL6 mice.At the end of drug treatment, left ventricular (LV) ejection fraction, fractional shortening and LV posterior wall thickness were not different in Apoe‐/‐ mice treated with either SKA‐31 or senicapoc compared with vehicle. Measurements of aortic PWV showed no significant differences among the three groups, which indicated that neither drug treatment decreased aortic stiffness. Oil‐Red‐O staining of the thoracic aorta and aortic arch revealed fatty plaque formation in Apoe‐/‐ mice (i.e. 10‐12% of area) compared with WT controls (< 1%), however, neither SKA‐31 nor senicapoc treatments reduced plaque formation vs. vehicle. Phenylephrine (PE)‐evoked contraction of abdominal aortic rings appeared similar in WT and vehicle/drug treated Apoe‐/‐ mice, whereas endothelium‐dependent, acetylcholine‐induced relaxation of PE‐constricted aortic rings was enhanced in aortic rings from mice treated with SKA‐31 and senicapoc vs. vehicle. Although relaxation to the smooth muscle vasodilator sodium nitroprusside was augmented in Apoe‐/‐ mice compared with WT, this response was not altered by drug treatment.While both SKA‐31 and senicapoc improved aortic endothelial function, neither drug decreased plaque formation or aortic stiffness, perhaps due to the later onset of these events in the progression of atherosclerotic pathology. We will investigate this likelihood by feeding Apoe‐/‐ mice a high fat diet to accelerate the development of plaque formation and arterial stiffness to determine the effects of both drug treatments on these later stages of atherosclerosis.

Prenatal stress enhances atherosclerosis and telomere shortening in ApoE knockout mouse offspring

Children born to women who experience stress during pregnancy have an increased risk of atherosclerosis in later life, but few animal models have explored mechanisms. To study this phenomenon, timed-bred ApoE knockout mice were determined pregnant with ultrasound and randomly assigned on gestation day 8.5 to either a control (no stress) or prenatal stress (PS) group using 2 h of restraint for five consecutive days. PS significantly increased plasma corticosterone levels in pregnant mice. The litters from PS mice showed increased neonatal mortality within the first week of life. Body weights (at euthanasia) of adult offspring at 25 wk from the PS group were significantly increased compared with weights of controls. Adult offspring from these pregnancies were serially imaged with ultrasound to measure plaque thickness and were compared with plaque macroscopic and microscopic pathology. PS groups had increased plaque thickness determined by ultrasound, gross, histological evaluation and increased aortic root and valve macrophage infiltration at 25 wk. Five-week-old mice from PS group had significant decrease in mean arterial pressure, yet blood pressure normalized by 10 wk. As prenatal stress induced increased atherosclerosis, and telomeres are susceptible to stress, aortas from 10-wk-old mice were compared for telomere lengths and were found to be significantly shorter in PS mice compared with control mice. These studies support future investigation of how stress impacts telomere shortening in animal models and human aortas. This model could be further used to investigate the role of prenatal stress, telomere biology, and atherosclerosis pathogenesis in adults.

Fibroblast growth factor 21 reverses high-fat diet-induced impairment of vascular function via the anti-oxidative pathway in ApoE knockout mice.

Circulating endothelial progenitor cells (EPCs), which function in vascular repair, are the markers of endothelial dysfunction and vascular health. Fibroblast growth factor 21 (FGF21), a liver‐secreted protein, plays a crucial role in glucose homeostasis and lipid metabolism. FGF21 has been reported to attenuate the progression of atherosclerosis, but its impact on EPCs under high oxidative stress conditions remains unclear. In vitro studies showed that the β‐klotho protein was expressed in cultured EPCs and that its expression was upregulated by FGF21 treatment. Hydrogen peroxide (H2O2)‐induced oxidative stress impaired EPC function, including cell viability, migration and tube formation. Pretreatment with FGF21 restored the functions of EPCs after the exposure to H2O2. Administration of N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME), an inhibitor of nitric oxide synthase, inhibited the effects of FGF21 in alleviating oxidative injury by suppressing endothelial nitric oxide synthase (eNOS). In an in vivo study, the administration of FGF21 significantly reduced total cholesterol (TC) and blood glucose levels in apolipoprotein E (ApoE)‐deficient mice that were fed a high‐fat diet (HFD). Endothelial function, as reflected by acetylcholine‐stimulated aortic relaxation, was improved after FGF21 treatment in ApoE‐deficient mice. Analysis of mRNA levels in the aorta indicated that FGF21 increased the mRNA expression of eNOS and upregulated the expression of the antioxidant genes superoxide dismutase (SOD)1 and SOD2 in ApoE‐deficient mice. These data suggest that FGF21 improves EPC functions via the Akt/eNOS/nitric oxide (NO) pathway and reverses endothelial dysfunction under oxidative stress. Therefore, administration of FGF21 may ameliorate a HFD‐induced vascular injury in ApoE‐deficient mice.

Endurance exercise ameliorates Western diet\u2013induced atherosclerosis through modulation of microbiota and its metabolites

The World Health Organization determined cardiovascular disease to be the leading cause of death globally; atherosclerosis is the primary cause of the high morbidity and mortality rates. Regular physical activity is an effective strategy for maintaining endothelial health and function to prevent the development of atherosclerosis. Obesity is also a crucial risk factor for atherosclerotic progression in combination with various complications and systemic inflammation. Physiological homeostasis is modulated by the intestinal microbiota, but the mechanisms through which exercise attenuates atherosclerosis through the microbiota have not been elucidated. Therefore, we investigated the effects of endurance exercise on atherosclerosis induced by a Western diet (WD) and apolipoprotein E (ApoE) knockout in terms of microbiota parameters and metabolites. Genetically modified ApoE knockout mice (C57BL/6-Apoeem1Narl/Narl, ApoEKO) and wild-type mice (C57BL6/J) were divided into the following four groups (n = 6), namely, wild-type mice fed a chow diet (WT CD), ApoEKO mice fed a chow diet (ApoE CD), ApoEKO mice fed a WD (ApoE WD), and ApoEKO mice fed a WD and performing endurance exercise (ApoE WD EX), for a 12-week intervention. The WD significantly induced obesity and atherosclerotic syndrome in the ApoE WD group. Severe atherosclerotic lesions and arterial thickness were significantly elevated and accompanied by increases in VCAM-1, MCP-1, TNF-α, and IL-1β for immune cell chemotaxis and inflammation during atherosclerotic pathogenesis in the ApoE WD group. In addition, dysbiosis in the ApoE WD group resulted in the lowest short-chain fatty acid (SCFA) production. Endurance exercise intervention (ApoE WD EX) significantly alleviated atherosclerotic syndrome by reducing obesity, significantly inhibiting VCAM-1, MCP-1, TNF-α, and IL-1β expression, and increasing the production of SCFAs. Modulation of the microbiota associated with inflammation, such as Desulfovibrio, Tyzzerella, and Lachnospiraceae_ge, and increased SCFA production, particularly through an abundance of Rikenellaceae and Dubosiella, were also observed after exercise intervention. Endurance exercise can alleviate WD-induced atherosclerosis through the amelioration of obesity, inflammation, and chemotaxis signaling, which are modulated by the microbiota and derived SCFAs.

Effects of 6-week aerobic exercise on calcium regulation in skeletal muscle of ApoE knockout mice fed by high-fat diet

Objective: To investigate the effects of 6 weeks of aerobic exercise on the sarcoplasmic reticulum calcium regulatory proteins in skeletal muscle of apolipoprotein E (ApoE) knockout mice fed by high-fat diet. Methods: There were a total of twenty five 9-week-old ApoE knockout mice (ApoE knockout mice, ApoE KO), five of which were selected randomly for the maximum running speed test. The running speed would be increased by 1.2 m/min every 3 min after a 5-min duration of initial speed of 4.8 m/min without slope until exhaustion, then the final speed was set as maximal speed, and the test result of the maximum running speed was (27.0±2.4)m/min. The remaining 20 ApoE KO mice were randomly divided into ApoE KO mouse high-fat diet group (KO) and ApoE KO mouse high-fat diet + aerobic exercise group (KE), 10 mice per group. Ten 9-week-old wild-type C57BL/6J mice were used as a blank control group (wild-type, WT). High fat diet composition: fat content was 21% (w/w) and cholesterol content was 1.5% (w/w). Exercise intervention was initiated 1 week after adaptive training in the KE group with an exercise protocol consisting of 40% maximal running speed (10.8 m/min), 40 min/d and a frequency of 3 d/w for a total of 6 weeks. All mice were anesthetized and sacrificed by cardiac puncture then bilateral gastrocnemius muscles were isolated immediately 48 h after the final exercise. Skeletal muscle Ca2+ concentration was detected by visible light colorimetry; the expression levels of RyR, CaM, CaMK Ⅱ, SERCA1 and SERCA2, which are calcium regulated proteins of sarcoplasmic reticulum, in mouse skeletal muscle, was detected by Western blotting. Results: Compared with WT mice, the Ca2+ concentration, the expression levels of the sarcoplasmic reticulum calcium releasing proteins RyR, CaMKⅡ, and the calcium recycling proteins SERCA1 and SERCA2 were decreased significantly in skeletal muscle of the KO mice (P＜0.05, P＜0.01), while the expression of CaM protein did not change. Skeletal muscle Ca2+ concentration and the levels of sarcoplasmic reticulum calcium recycling proteins SERCA1 and SERCA2 were increased significantly (P＜0.05) in KE mice compared with KO mice, but there were no significant differences in the expressions of the sarcoplasmic reticulum calcium release proteins RyR, CaM and CaMK II. Conclusion: High fat diet can reduce the concentration of Ca2+ in skeletal muscle and weaken the release and recovery of sarcoplasmic reticulum calcium in ApoE knockout mice. 6-week aerobic exercise training can significantly increase its Ca2+concentration and promote the recovery of sarcoplasmic reticulum calcium.

Tolvaptan reduces angiotensin II-induced experimental abdominal aortic aneurysm and dissection

Tolvaptan has been approved for the treatment of autosomal dominant polycystic kidney disease and heart failure. However, the role of tolvaptan in patients with abdominal aortic aneurysm (AAA) has not been examined. Human aortic smooth muscle cells (HASMCs) were used as the in vitro model. Via Ang II infusion, experimental AAAs were induced in Apo-E knockout mice. In vitro study showed that tolvaptan suppressed matrix metalloproteinase (MMP) expressions (MMP-2 and MMP-9) and apoptosis in Ang II-stimulated HASMCs. In the Apo-E knockout mice with Ang II-induced AAA, the animals exhibited AAA formation with elastic lamina degradation, dilatation of the suprarenal aorta, increased macrophage infiltration and higher expressions of MMPs. Treatment with a high dose of tolvaptan prevented experimental AAA formation while preserving the elastic lamina structure, reducing inflammatory macrophages, and inhibiting gelatinolytic activity, MMP expressions and apoptosis of SMCs in aorta tissue. Specifically, tolvaptan reduced the expression of receptor-interacting protein kinase 3 (RIP3) and decreased apoptosis of SMCs. Our data demonstrated that tolvaptan reduces experimental AAA formation and dissection by inhibiting destruction of the aortic structure integrity and reducing inflammatory macrophage infiltration, MMP-2 and MMP-9 expressions, and apoptosis of vascular SMCs, indicating tolvaptan may have therapeutic potential for AAA and dissection.

Effects of exercise on high-fat diet\u2013induced non-alcoholic fatty liver disease and lipid metabolism in ApoE knockout mice

BackgroundNon-alcoholic fatty liver disease (NAFLD), which is growing more common in the Western world, has become the main cause of chronic liver disease and is strongly associated with metabolism syndromes. NAFLD can indicate a wide spectrum of hepatic pathologies, ranging from simple hepatic steatosis and inflammatory non-alcoholic steatohepatitis to more severe stages of fibrosis and cirrhosis. Moreover, evidence has demonstrated that physical inactivity and westernized dietary habits may facilitate the development of NAFLD. Lipid modulation and metabolism could be important factors in the development of steatosis. Lipid species, characterized using a lipidomic approach with untargeted analysis, could provide potential biomarkers for the pathogenesis of NAFLD or therapeutic applications. Thus, in this study, the effects of exercise on the improvement of NAFLD were further investigated from a lipidomic perspective through the aspects of lipid regulation and metabolism.MethodsWild type (WT) C57BL/6 J and C57BL/6-ApoEem1Narl/Narl mice were assigned to one of four groups: WT mice fed a normal chow diet (CD), apolipoprotein E (ApoE) knockout mice fed a normal CD, ApoE knockout mice fed a high-fat diet (HFD), and ApoE knockout mice fed a HFD and provided with swimming exercise. The treatments (e.g., normal diet, HFD, and exercise) were provided for 12 consecutive weeks before the growth curves, biochemistry, fat composition, pathological syndromes, and lipid profiles were determined.ResultsExercise significantly reduced the HFD-induced obesity (weight and fat composition), adipocyte hypertrophy, liver lipid accumulation, and pathological steatosis. In addition, exercise ameliorated HFD-induced steatosis in the process of NAFLD. The lipidomic analysis revealed that the changes in plasma triglyceride (14:0/16:0/22:2), phosphatidic acid (18:0/17:2), and phosphatidylglycerol (16:0/20:2) induced by the administration of the HFD could be reversed significantly by exercise.ConclusionsThe 12-week regular exercise intervention significantly alleviated HFD-induced NAFLD through modulation of specific lipid species in plasma. This finding could elucidate the lipids effects behind the hepatic pathogenesis with exercise.

TCDD aggravates the formation of the atherosclerotic plaque in ApoE KO mice with a sexual dimorphic pattern

Aryl hydrocarbon receptor (AhR) ligands are recognized as aggravating factors in cardiovascular diseases but little is known about the role of the AhR in atherosclerosis considering the effects of age and gender. We exposed male and female ApoE knock-out mice, a model to study the pathogenesis of atherosclerosis, to a potent AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by an intraperitoneal injection of 1 μg/kg/week for 8 weeks. Atherosclerotic lesions, histological parameters and critical atherosclerotic markers in aorta were analysed. TCDD increased atherogenic lesions in 35-week old female mice, leading to a switch of vascular smooth muscle cells (VSMCs) from a contractile to a pro-atherogenic phenotype and increased expression for VCAM1. AhR activation accelerates the formation of atherosclerotic plaques with sex and age differences due to the phenotypical switch of VSMCs.

Estrogen Sulfotransferase is Highly Expressed in Vascular Endothelial Cells Overlying Atherosclerotic Plaques.

Cytosolic estrogen sulfotransferase (SULT1E1) mainly catalyzes the sulfoconjugation and deactivation of estrogens that are known to exert potent anti-atherogenic effects. However, it remains unknown about the connection between SULT1E1 andatherosclerosis. Recently, we reported that SULT1E1 is highly expressed in the aorta with plaques of high fat-fed ApoE knockout (KO) mice (mouse model of atherosclerosis), and interacts with oxidized low-density lipoprotein (Ox-LDL) known as a major component of atherosclerotic lesions. In this study, immunohistochemical staining for SULT1E1 in the aorta of high fat-fed ApoE KO mice showed that SULT1E1 is detected in vascular endothelial cells overlying atherosclerotic plaques. Results from Western blotting showed that Ox-LDL induces the protein expression of both SULT1E1 and peroxisome proliferator-activated receptor (PPAR) γ in human umbilical vein endothelial cells (HUVECs), and then that a PPARγ antagonist GW9662, but not a PPARα antagonist GW6471, inhibited the protein expression of SULT1E1 induced by Ox-LDL. Moreover, GW9662 significantly increased the proliferation of HUVECs induced by Ox-LDL. Our results suggest that SULT1E1 and PPARγ, both of which are increased by Ox-LDL, may interact with each other, and then may reduce cooperatively Ox-LDL-induced proliferation of vascular endothelial cells overlying atherosclerotic plaques, leading to against atherosclerosis.

Protective role for calcitonin gene-related peptide in atherosclerosis progress in ApoE knockout mice

Protective role for calcitonin gene-related peptide in atherosclerosis progress in ApoE knockout mice

Neurotoxic 18-kDa apolipoprotein E fragment production contributes to anesthetic sevoflurane-induced tau phosphorylation and neuroinflammation in vitro.

Anesthesia may induce neuronal tau phosphorylation and neurotoxicity in the developing brain. Apolipoprotein E (ApoE) may play a protective role in neuronal activity and injury repair, whereas its 18-kDa fragments are reported to induce neurodegeneration and neuroinflammation in Alzheimer's disease patients. We aimed to test the hypothesis that differences in 18-kDa ApoE fragment levels, but not full-length ApoE, in primary neurons contribute to differences in tau phosphorylation and neuroinflammation with or without sevoflurane administration. Neurons extracted from wild-type (WT), ApoE knockout (ApoE-KO), and ApoE ε3-and ε2-targeted replacement (ApoE ε3, ApoE ε2) mice were divided into control and sevoflurane groups. Neurons in the sevoflurane group were treated with 21% O2, 5% CO2, and 4.1% sevoflurane, whereas those in the control group were treated with 21% O2 and 5% CO2 only on day 5 of neuronal culture. ApoE mRNA, full-length ApoE, 18-kDa ApoE fragments, Tau-PS202/PT205 (AT8), Tau-PSer396/404 (PHF1), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 and IL-1β levels were measured. The data showed that sevoflurane-induced AT8 and PHF1 increases, and TNF-α, IL-6, and IL-1β increases in WT or ApoE ε3 neurons (both expressing full-length and 18-kDa fragmented ApoE) could be mitigated in ApoE ε2 (only expressing full-length ApoE), but not in ApoE-KO neurons, indicating that differences in 18-kDa ApoE fragments, but not full-length ApoE, in primary mouse neurons contributed to differences in tau phosphorylation and neuroinflammation with or without 4.1% sevoflurane administration.

Apolipoprotein E loss of function: Influence on murine brain markers of physiology and pathology.

The canonical role of Apolipoprotein E (ApoE) is related to lipid and cholesterol metabolism, however, additional functions of this protein have not been fully described. Given the association of ApoE with diseases such as Alzheimer's Disease (AD), it is clear that further characterisation of its roles, especially within the brain, is needed. Therefore, using protein and gene expression analyses of neonatal and 6-month old brain tissues from an ApoE knockout mouse model, we examined ApoE's contribution to several CNS pathways, with an emphasis on those linked to AD. Early neonatal changes associated with ApoE-/- were observed, with decreased soluble phosphorylated tau (p-tau, -40 %), increased synaptophysin (+36 %) and microglial Iba1 protein levels (+25 %) vs controls. Progression of the phenotype was evident upon analysis of 6-month-old tissue, where decreased p-tau was also confirmed in the insoluble fraction, alongside reduced synaptic and increased amyloid precursor protein (APP) protein levels. An age comparison further underlined deviations from WT animals and thus the impact of ApoE loss on neuronal maturation. Taken together, our data implicate ApoE modulation of multiple CNS roles. Loss of function is associated with alterations from birth, and include synaptic deficits, neuroinflammation, and changes to key AD pathologies, amyloid-β and tau.

Autophagy enhanced by curcumin ameliorates inflammation in atherogenesis via the TFEB–P300–BRD4 axis

Disturbance of macrophage-associated lipid metabolism plays a key role in atherosclerosis. Crosstalk between autophagy deficiency and inflammation response in foam cells (FCs) through epigenetic regulation is still poorly understood. Here, we demonstrate that in macrophages, oxidized low-density lipoprotein (ox-LDL) leads to abnormal crosstalk between autophagy and inflammation, thereby causing aberrant lipid metabolism mediated through a dysfunctional transcription factor EB (TFEB)–P300–bromodomain-containing protein 4 (BRD4) axis. ox-LDL led to macrophage autophagy deficiency along with TFEB cytoplasmic accumulation and increased reactive oxygen species generation. This activated P300 promoted BRD4 binding on the promoter regions of inflammatory genes, consequently contributing to inflammation with atherogenesis. Particularly, ox-LDL activated BRD4-dependent super-enhancer associated with liquid–liquid phase separation (LLPS) on the regulatory regions of inflammatory genes. Curcumin (Cur) prominently restored FCs autophagy by promoting TFEB nuclear translocation, optimizing lipid catabolism, and reducing inflammation. The consequences of P300 and BRD4 on super-enhancer formation and inflammatory response in FCs could be prevented by Cur. Furthermore, the anti-atherogenesis effect of Cur was inhibited by macrophage-specific Brd4 overexpression or Tfeb knock-out in Apoe knock-out mice via bone marrow transplantation. The findings identify a novel TFEB-P300-BRD4 axis and establish a new epigenetic paradigm by which Cur regulates autophagy, inhibits inflammation, and decreases lipid content.

Deletion of ApoE Leads to Intervertebral Disc Degeneration via Aberrant Activation of Adipokines.

Animal experiment: a mouse model of intervertebral disc (IVD) degeneration induced by deletion of apolipoprotein E (apoE). The aim of this study was to investigate the role and mechanism of apoE on the process of IVD degeneration. Abnormal lipid metabolism has been demonstrated to be closely related to IVD degeneration, a common chronic degenerative joint disease. ApoE, a component of apolipoproteins, plays a crucial role in lipid transportation and metabolic balance. But the relationship between apoE and IVD degeneration remains largely unknown. ApoE knockout (KO) mouse was employed to investigate the progressive disc degeneration. The changes of vertebral bone and intervertebral disc space were measured by micro-computed tomography (micro-CT). The histo-morphological changes of cartilage endplate (CEP) and underlying signals were tested using immunohistochemistry and immunofluorescence staining. The deletion of apoE gene accelerated the lumbar spine degeneration. Compared with WT mice, apoE KO mice showed reduced IVD space and increased vertebral bone mass. The progressive CEP degeneration was further found with cartilage degradation and endplate sclerosis in apoE KO mice. The deletion of apoE stimulated abnormal CEP bone remodeling and activation of adipokines signals. The deletion of apoE gene induced abnormal activation of adipokines signals, thus contribute to the CEP degeneration. N/A.

Tanyu Tongzhi Formula Delays Atherosclerotic Plaque Progression by Promoting Alternative Macrophage Activation via PPARγ and AKT/ERK Signal Pathway in ApoE Knock-Out Mice.

We previously demonstrated that the Tanyu Tongzhi Formula (TTF) significantly alleviated the clinical symptoms of patients with coronary heart disease and lowered serum lipid and inflammatory factor levels in patients with coronary heart disease and atherosclerosis model rats. However, the mechanism underlying TTF remains unknown. In this study, we examined the effect of TTF on atherosclerotic plaques in ApoE-/- mice and underlying mechanisms involved in macrophage polarization. Sixty male ApoE-/- mice were randomly divided into four groups. Mice in the control group were fed a regular diet, whereas experimental mice were fed a high-fat diet and received either saline (HFD group) or TTF at concentrations of 0.60 (TTF-L group) or 2.25 g/ml (TTF-H group) by daily oral gavage for 16 weeks. In the TTF-L and TTF-H groups, the levels of serum cholesterol, triglyceride, interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were decreased, lipid content was significantly decreased, and percentage area of collagen/lipid increased in atherosclerotic plaque compared to in the HFD group. Moreover, we found TTF promoted the expression of alternative macrophage markers (Fizz1, Arg1, and Mrc) and suppressed the expression of M1 macrophage markers (TNF-α, IL-1β, and IL-6) by regulating peroxisome proliferator-activated receptor γ (PPARγ) expression and AKT/extracellular signal-regulated kinase (ERK) activation. We further investigated whether alternative macrophage was reduced when PPARγ was inhibited or the AKT/ERK signaling pathway was activated. TTF delayed atherosclerotic plaque progression by promoting alternative macrophage activation through increasing PPARγ expression and inhibiting AKT/ERK phosphorylation, providing a theoretical basis for its clinical application.

Artesunate attenuates atherosclerosis by inhibiting macrophage M1-like polarization and improving metabolism

ObjectAtherosclerosis (AS) is caused by chronic inflammation. Artesunate (ART), a sesquiterpene lactone endoperoxide isolated from Chinese herbal medicine, displays excellent anti-inflammatory activity. In this study, we investigated the effects of artesunate on atherosclerosis in ApoE knock-out mice, and used untargeted metabolomics to determine metabolite changes in these mice following ART treatment. MethodsApoE knock-out mice were fed a western diet and administered ART for eight weeks. Untargeted metabolomics was used to detect differential metabolites following the administration of ART. Oil Red O was used to assess plaque size, western blot and ELISA were used to detect inflammatory factors, and flow cytometry was used to detect the expression of markers on macrophages. ResultsResults of the in vivo experiment suggested that ART reduced atherosclerotic plaques in murine aortic root. In addition both in vivo and vitro experiments suggested that ART reduced the expression levels of inflammating cytokines, but enhanced those of the anti-inflammatory cytokines in macrophages. Untargeted metabolomic analysis demonstrated that multiple metabolic pathways, which were blocked in AS mice, showed different degrees of improvement following ART treatment. Furthermore, bioinformatic analyses showed that the HIF-1α pathway was altered in the AS mice and the ART treatment mice. In vitro experiments confirmed that LPS-induced upregulation of HIF-1α expression and activation of the NF-κB signaling pathways was significantly inhibited by ART treatment. ConclusionThese results suggest that ART exerts anti-atherosclerosis effects by inhibiting M1 macrophage polarization. One of the molecular mechanisms is that ART inhibits M1-like macrophage polarization via regulating HIF-1α and NF-κB signaling pathways.