A novel mechanism of ferroptosis inhibition-enhanced atherosclerotic plaque stability: YAP1 suppresses vascular smooth muscle cell ferroptosis through GLS1.

Atherosclerosis is a leading cause of cardiovascular diseases (CVDs), often resulting in major adverse cardiovascular events (MACEs), such as myocardial infarction and stroke due to the rupture or erosion of vulnerable plaques. Ferroptosis, an iron-dependent form of cell death, has been implicated in the development of atherosclerosis. Despite its involvement in CVDs, the specific role of ferroptosis in atherosclerotic plaque stability remains unclear. In this study, we confirmed the presence of ferroptosis in unstable atherosclerotic plaques and demonstrated that the ferroptosis inhibitor ferrostatin-1 (Fer-1) stabilizes atherosclerotic plaques in apolipoprotein E knockout (Apoe-/-) mice. Using bioinformatic analysis combining RNA sequencing (RNA-seq) with single-cell RNA sequencing (scRNA-seq), we identified Yes-associated protein 1 (YAP1) as a potential key regulator of ferroptosis in vascular smooth muscle cells (VSMCs) of unstable plaques. In vitro, we found that YAP1 protects against oxidized low-density lipoprotein (oxLDL)-induced ferroptosis in VSMCs. Mechanistically, YAP1 exerts its anti-ferroptosis effects by regulating the expression of glutaminase 1 (GLS1) to promote the synthesis of glutamate (Glu) and glutathione (GSH). These findings establish a novel mechanism where the inhibition of ferroptosis promotes the stabilization of atherosclerotic plaques through the YAP1/GLS1 axis, attenuating VSMC ferroptosis. Thus, targeting the YAP1/GLS1 axis to suppress VSMC ferroptosis may represent a novel strategy for preventing and treating unstable atherosclerotic plaques.

Methyl-CpG-binding 2 K271 lactylation-mediated M2 macrophage polarization inhibits atherosclerosis.

Rationale: Posttranslational modifications of proteins have not been addressed in studies aimed at elucidating the cardioprotective effect of exercise in atherosclerotic cardiovascular disease (ASCVD). In this study, we reveal a novel mechanism by which exercise ameliorates atherosclerosis via lactylation. Methods: Using ApoE-/- mice in an exercise model, proteomics analysis was used to identify exercise-induced specific lactylation of MeCP2 at lysine 271 (K271). Mutation of the MeCP2 K271 lactylation site in aortic plaque macrophages was achieved by recombinant adenoviral transfection. Explore the molecular mechanisms by which motility drives MeCP2 K271 lactylation to improve plaque stability using ATAC-Seq, CUT &Tag and molecular biology. Validation of the potential target RUNX1 for exercise therapy using Ro5-3335 pharmacological inhibition. Results: we showed that in ApoE-/- mice, methyl-CpG-binding protein 2 (MeCP2) K271 lactylation was observed in aortic root plaque macrophages, promoting pro-repair M2 macrophage polarization, reducing the plaque area, shrinking necrotic cores, reducing plaque lipid deposition, and increasing collagen content. Adenoviral transfection, by introducing a mutant at lysine 271, overexpressed MeCP2 K271 lactylation, which enhanced exercise-induced M2 macrophage polarization and increased plaque stability. Mechanistically, the exercise-induced atheroprotective effect requires an interaction between MeCP2 K271 lactylation and H3K36me3, leading to increased chromatin accessibility and transcriptional repression of RUNX1. In addition, the pharmacological inhibition of the transcription factor RUNX1 exerts atheroprotective effects by promoting the polarization of plaque macrophages towards the pro-repair M2 phenotype. Conclusions: These findings reveal a novel mechanism by which exercise ameliorates atherosclerosis via MeCP2 K271 lactylation-H3K36me3/RUNX1. Interventions that enhance MeCP2 K271 lactylation have been shown to increase pro-repair M2 macrophage infiltration, thereby promoting plaque stabilization and reducing the risk of atherosclerotic cardiovascular disease. We also established RUNX1 as a potential drug target for exercise therapy, thereby providing guidance for the discovery of new targets.

LRG1 promotes atherosclerosis by inducing macrophage M1-like polarization.

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by the accumulation of cholesterol-rich lipoproteins in macrophages. How macrophages commit to proinflammatory polarization under atherosclerosis conditions is not clear. Report here that the level of a circulating protein, leucine-rich alpha-2 glycoprotein 1 (LRG1), is elevated in the atherosclerotic tissue and serum samples from patients with coronary artery disease (CAD). LRG1 stimulated macrophages to proinflammatory M1-like polarization through the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways. The LRG1 knockout mice showed significantly delayed atherogenesis progression and reduced levels of macrophage-related proinflammatory cytokines in a high-fat diet-induced Apoe-/- mouse atherosclerosis model. An anti-LRG1 neutralizing antibody also effectively blocked LRG1-induced macrophage M1-like polarization in vitro and conferred therapeutic benefits to animals with ApoE deficiency-induced atherosclerosis. LRG1 may therefore serve as an additional biomarker for CAD and targeting LRG1 could offer a potential therapeutic strategy for CAD patients by mitigating the proinflammatory response of macrophages.

Aberrant mitochondrial DNA synthesis in macrophages exacerbates inflammation and atherosclerosis.

There is a large body of evidence that cellular metabolism governs inflammation, and that inflammation contributes to the progression of atherosclerosis. However, whether mitochondrial DNA synthesis affects macrophage function and atherosclerosis pathology is not fully understood. Here we show, by transcriptomic analyzes of plaque macrophages, spatial single cell transcriptomics of atherosclerotic plaques, and functional experiments, that mitochondrial DNA (mtDNA) synthesis in atherosclerotic plaque macrophages are triggered by vascular cell adhesion molecule 1 (VCAM-1) under inflammatory conditions in both humans and mice. Mechanistically, VCAM-1 activates C/EBPα, which binds to the promoters of key mitochondrial biogenesis genes - Cmpk2 and Pgc1a. Increased CMPK2 and PGC-1α expression triggers mtDNA synthesis, which activates STING-mediated inflammation. Consistently, atherosclerosis and inflammation are less severe in Apoe-/- mice lacking Vcam1 in macrophages. Downregulation of macrophage-specific VCAM-1 in vivo leads to decreased expression of LYZ1 and FCOR, involved in STING signalling. Finally, VCAM-1 expression in human carotid plaque macrophages correlates with necrotic core area, mitochondrial volume, and oxidative damage to DNA. Collectively, our study highlights the importance of macrophage VCAM-1 in inflammation and atherogenesis pathology and proposes a self-acerbating pathway involving increased mtDNA synthesis.

APOE-NOTCH axis governs elastogenesis during human cardiac valve remodeling.

Valve remodeling is a process involving extracellular matrix organization and elongation of valve leaflets. Here, through single-cell RNA sequencing of human fetal valves, we identified an elastin-producing valve interstitial cell (VIC) subtype (apolipoprotein E (APOE)+, elastin-VICs) spatially located underneath valve endothelial cells (VECs) sensing unidirectional flow. APOE knockdown in fetal VICs resulted in profound elastogenesis defects. In valves with pulmonary stenosis (PS), we observed elastin fragmentation and decreased expression of APOE along with other genes regulating elastogenesis. Cell-cell interaction analysis revealed that jagged 1 (JAG1) from unidirectional VECs activates elastogenesis in elastin-VICs through NOTCH2. Similar observations were made in VICs cocultured with VECs under unidirectional flow. Notably, a drastic reduction of JAG1-NOTCH2 was also observed in PS valves. Lastly, we found that APOE controls JAG1-induced NOTCH activation and elastogenesis in VICs through the extracellular signal-regulated kinase pathway. Our study suggests important roles of both APOE and NOTCH in regulating elastogenesis during human valve remodeling.

Huanglian Jiedu Decoction enhances the stability of atherosclerotic plaques through SLC2A1-mediated efferocytosis.

BACKGROUND: Atherosclerotic (AS) plaques require a dense necrotic core and a robust fibrous cap to maintain stability. While previous studies have indicated that the traditional Chinese medicine Huang Lian Jie Du Decoction (HLJDD) possesses the capability to stabilize AS plaques, the underlying mechanisms remain obscure. This study aims to delve deeper into the potential mechanisms by which HLJDD improves AS through an integrated research strategy. METHODS: Leveraging an AS model in ApoE-/- mice exposed to a high-fat diet (HFD), we scrutinized the therapeutic effects of HLJDD using microscopic observations, oil red O staining, HE staining and Masson staining. Employing comprehensive techniques of network pharmacology, bioinformatics, and molecular docking, we elucidated the mechanism by which HLJDD stabilizes AS plaques. In vitro experiments, utilizing ox-LDL-induced macrophages and apoptotic vascular smooth muscle cells (VSMCs), assessed the impact of HLJDD on efferocytosis and the role of SLC2A1. RESULTS: In vivo experiments showcased the efficacy of HLJDD in reducing the quantity of aortic plaques, diminishing lipid deposition, and enhancing plaque stability in AS mice. Employing network pharmacology and machine learning, we pinpointed SLC2A1 as a crucial regulatory target. Molecular docking further validated the binding of HLJDD components with SLC2A1. The experiments demonstrated a dose-dependent upregulation in SLC2A1 expression by HLJDD, amplifying efferocytosis. Importantly, this effect was reversed by the SLC2A1 inhibitor STF-31, highlighting the pivotal role of SLC2A1 as a target. CONCLUSION: The HLJDD can modulate macrophage efferocytosis by enhancing the expression levels of SLC2A1, thereby improving the stability of atherosclerotic plaques.

TMAO induces pyroptosis of vascular endothelial cells and atherosclerosis in ApoE-/- mice via MBOAT2-mediated endoplasmic reticulum stress.

Trimethylamine N-oxide (TMAO), a metabolite produced by intestinal flora, is recognized as an independent risk factor for atherosclerosis and atherosclerotic cardiovascular diseases. However, the underlying mechanism remains poorly understood. Here, we showed that dietary TMAO supplementation accelerates atherosclerosis in ApoE-/- mice. Pyroptosis and the expression of phospholipid-modifying enzyme MBOAT2 were increased in endothelial cells within atherosclerotic lesions. Genetic upregulation of MBOAT2 via adeno-associated virus with endothelium-specific promoter results in increased atherosclerotic lesions in ApoE-/- mice. Mechanistically, the overexpression of MBOAT2 disrupted glycerophospholipid metabolism and induced endothelial cell pyroptosis in an Endoplasmic reticulum stress-dependent manner. These data reveal that TMAO promotes endothelial cell pyroptosis and the progression of atherosclerotic lesions through the upregulation of MBOAT2, indicating that MBOAT2 is a promising therapeutic target for atherosclerosis.

[Imaging for displaying lymphatic vessels in whole-mount aorta adventitia of ApoE-/- mice by immunofluorescent histochemical staining].

Objective To explore a simple and feasible method for whole-mount immunofluorescence staining of lymphatic vessels in the ApoE-/- mouse model of atherosclerosis. Methods Aortic specimens were carefully excised from the ApoE-/- mouse model. Following immunostaining with specific antibodies against smooth muscle actin (SMA) and lymphatic vessel endothelial receptor 1 (LYVE1), the aortas, including the aortic root, were subjected to a 30-minute treatment with 5 g/L Sudan Black B solution. This step was instrumental in minimizing the autofluorescent background of the tissue. Thereafter, the aortas were processed through a clearing protocol and imaged within a purpose-built chamber under a fluorescence microscope. Results The pretreatment with 5 g/L Sudan Black B effectively suppressed the autofluorescent signals emanating from the vascular structures, thereby enhancing the contrast and clarity of the specific fluorescence signals associated with the lymphatic vessels. This enhancement in signal quality did not compromise the integrity or specificity of the immunofluorescent markers. Conclusion A facile, highly specific, and effective approach for the visualization of lymphatic vessels in whole-mount aortic preparations from ApoE-/- mice is established.

Exacerbation of atherosclerosis by STX17 knockdown: Unravelling the role of autophagy and inflammation.

Syntaxin 17 (STX17) has been identified as a crucial factor in mediating the fusion of autophagosomes and lysosomes. However, its specific involvement in the context of atherosclerosis (AS) remains unclear. This study sought to elucidate the role and mechanistic contributions of STX17 in the initiation and progression of AS. Utilizing both in vivo and in vitro AS model systems, we employed ApoE knockout (KO) mice subjected to a high-fat diet and human umbilical vein endothelial cells (HUVECs) treated with oxidized low-density lipoprotein (ox-LDL) to assess STX17 expression. To investigate underlying mechanisms, we employed shRNA-STX17 lentivirus to knock down STX17 expression, followed by evaluating autophagy and inflammation in HUVECs. In both in vivo and in vitro AS models, STX17 expression was significantly upregulated. Knockdown of STX17 exacerbated HUVEC damage, both with and without ox-LDL treatment. Additionally, we observed that STX17 knockdown impaired autophagosome degradation, impeded autophagy flux and also resulted in the accumulation of dysfunctional lysosomes in HUVECs. Moreover, STX17 knockdown intensified the inflammatory response following ox-LDL treatment in HUVECs. Further mechanistic exploration revealed an association between STX17 and STING; reducing STX17 expression increased STING levels. Further knockdown of STING enhanced autophagy flux. In summary, our findings suggest that STX17 knockdown worsens AS by impeding autophagy flux and amplifying the inflammatory response. Additionally, the interaction between STX17 and STING may play a crucial role in STX17-mediated autophagy.

Melatonin Ameliorates Atherosclerotic Plaque Vulnerability by Regulating PPARδ-Associated Smooth Muscle Cell Phenotypic Switching.

Vulnerable atherosclerotic plaque rupture, the leading cause of fatal atherothrombotic events, is associated with an increased risk of mortality worldwide. Peroxisome proliferator-activated receptor delta (PPARδ) has been shown to modulate vascular smooth muscle cell (SMC) phenotypic switching, and, hence, atherosclerotic plaque stability. Melatonin reportedly plays a beneficial role in cardiovascular diseases; however, the mechanisms underlying improvements in atherosclerotic plaque vulnerability remain unknown. In this study, we assessed the role of melatonin in regulating SMC phenotypic switching and its consequential contribution to the amelioration of atherosclerotic plaque vulnerability and explored the mechanisms underlying this process. We analyzed features of atherosclerotic plaque vulnerability and markers of SMC phenotypic transition in high-cholesterol diet (HCD)-fed apolipoprotein E knockout (ApoE-/-) mice and human aortic SMCs (HASMCs). Melatonin reduced atherosclerotic plaque size and necrotic core area while enhancing collagen content, fibrous cap thickness, and smooth muscle alpha-actin positive cell coverage on the plaque cap, which are all known phenotypic characteristics of vulnerable plaques. In atherosclerotic lesions, melatonin significantly decreased the synthetic SMC phenotype and KLF4 expression and increased the expression of PPARδ, but not PPARα and PPARγ, in HCD-fed ApoE-/- mice. These results were subsequently confirmed in the melatonin-treated HASMCs. Further analysis using PPARδ silencing and immunoprecipitation assays revealed that PPARδ plays a role in the melatonin-induced SMC phenotype switching from synthetic to contractile. Collectively, we provided the first evidence that melatonin mediates its protective effect against plaque destabilization by enhancing PPARδ-mediated SMC phenotypic switching, thereby indicating the potential of melatonin in treating atherosclerosis.

Quercetin Attenuates KLF4-Mediated Phenotypic Switch of VSMCs to Macrophage-like Cells in Atherosclerosis: A Critical Role for the JAK2/STAT3 Pathway.

The objective of this study was to elucidate the protective role of quercetin in atherosclerosis by examining its effect on the phenotypic switch of vascular smooth muscle cells (VSMCs) to macrophage-like cells and the underlying regulatory pathways. Aorta tissues from apolipoprotein E-deficient (ApoE KO) mice fed a high-fat diet (HFD), treated with or without 100 mg/kg/day quercetin, were analyzed for histopathological changes and molecular mechanisms. Quercetin was found to decrease the size of atherosclerotic lesions and mitigate lipid accumulation induced by HFD. Fluorescence co-localization analysis revealed a higher presence of macrophage-like vascular smooth muscle cells (VSMCs) co-localizing with phospho-Janus kinase 2 (p-JAK2), phospho-signal transducer and activator of transcription 3 (p-STAT3), and Krüppel-like factor 4 (KLF4) in regions of foam cell aggregation within aortic plaques. However, this co-localization was reduced following treatment with quercetin. Quercetin treatment effectively inhibited the KLF4-mediated phenotypic switch in oxidized low-density lipoprotein (ox-LDL)-loaded mouse aortic vascular smooth muscle cells (MOVAS), as indicated by decreased expressions of KLF4, LGALS3, CD68, and F4/80, increased expression of alpha smooth muscle actin (α-SMA), reduced intracellular fluorescence Dil-ox-LDL uptake, and decreased lipid accumulation. In contrast, APTO-253, a KLF4 activator, was found to reverse the effects of quercetin. Furthermore, AG490, a JAK2 inhibitor, effectively counteracted the ox-LDL-induced JAK2/STAT3 pathway-dependent switch to a macrophage-like phenotype and lipid accumulation in MOVAS cells. These effects were significantly mitigated by quercetin but exacerbated by coumermycin A1, a JAK2 activator. Our research illustrates that quercetin inhibits the KLF4-mediated phenotypic switch of VSMCs to macrophage-like cells and reduces atherosclerosis by suppressing the JAK2/STAT3 pathway.

APOE4 Increases Energy Metabolism in APOE-Isogenic iPSC-Derived Neurons.

The apolipoprotein E4 (APOE4) allele represents the major genetic risk factor for Alzheimer's disease (AD). In contrast, APOE2 is known to lower the AD risk, while APOE3 is defined as risk neutral. APOE plays a prominent role in the bioenergetic homeostasis of the brain, and early-stage metabolic changes have been detected in the brains of AD patients. Although APOE is primarily expressed by astrocytes in the brain, neurons have also been shown as source for APOE. However, the distinct roles of the three APOE isoforms in neuronal energy homeostasis remain poorly understood. In this study, we generated pure human neurons (iN cells) from APOE-isogenic induced pluripotent stem cells (iPSCs), expressing either APOE2, APOE3, APOE4, or carrying an APOE knockout (KO) to investigate APOE isoform-specific effects on neuronal energy metabolism. We showed that endogenously produced APOE4 enhanced mitochondrial ATP production in APOE-isogenic iN cells but not in the corresponding iPS cell line. This effect neither correlated with the expression levels of mitochondrial fission or fusion proteins nor with the intracellular or secreted levels of APOE, which were similar for APOE2, APOE3, and APOE4 iN cells. ATP production and basal respiration in APOE-KO iN cells strongly differed from APOE4 and more closely resembled APOE2 and APOE3 iN cells, indicating a gain-of-function mechanism of APOE4 rather than a loss-of-function. Taken together, our findings in APOE isogenic iN cells reveal an APOE genotype-dependent and neuron-specific regulation of oxidative energy metabolism.

Generation of induced pluripotent stem cell line (XWHNi003-A) from a female with APOE gene mutation.

Apolipoprotein E (APOE)is the gene with greatest genetic risk for Alzheimer's disease (AD). We successfully established a human induced pluripotent stem cell(iPSC) line from a woman mutated by APOE gene. The cell line was isolated from this woman's peripheral blood mononuclear cells using a non-integrated Sendai virus, which retained the original genotype, showed a normal karyotype, highly expressed pluripotent markers and could differentiate into three germ layers.

Low-dose radiation promotes high-fat diet-induced atherosclerosis by activating cGAS signal pathway.

BACKGROUND: Atherosclerosis (AS) is the most prevalent cardiovascular disease, with an exceptionally high burden. High-fat diet (HFD) is a popular diet behavior, whereas low-dose radiation (LDR) is an environmental physical factor. There is evidence to suggest that an HFD may exacerbate the onset of atherosclerosis. Whether the combination effect of HFD and LDR would have potential on atherosclerosis development remains incompletely unclear. METHODS: In this study, ApoE-/- mice were used as atherosclerosis model animals to investigate the combination effects of HFD and LDR (10 × 0.01Gy, or 20 × 0.01Gy) on vascular lesions. Doppler ultrasound imaging, H&E staining, oil red O staining, western blotting, and immunohistochemistry (IHC) were used to assess the pro-atherosclerotic effects. LC-MS was used to detect the non-targeted lipidomic. RESULTS: Long-term exposure of low-dose radiation at an accumulated dose of 0.2Gy significantly increased the occurrence of vascular stiffness and the aortic lesion in ApoE-/- mice. The synergistic effect of HFD and LDR was observed in the development of atherosclerosis, which might be linked to both the dysbiosis of lipid metabolism and the stimulation of the inflammatory signaling system. Moreover, LDR but not HFD can activate the cGAS-STING signaling through increasing the yield of cytosolic mitochondrial DNAs as well as the expression of cGAS protein. The activation of cGAS-STING signal triggers the release of IFN-α/-ß, which functions as an inflammatory amplifier in the formation of atherosclerotic plaque. CONCLUSION: The current study offers fresh insights into the risks and mechanism that underlie the development of atherosclerosis by LDR, and there is a combination effect of LDR and HFD with the involvement of cGAS-STING signal pathway.

Evaluation of the anti-atherosclerotic effect for Allium macrostemon Bge. Polysaccharides and structural characterization of its a newly active fructan.

Allium Macrostemon Bge. (AMB) is a well-known homology of herbal medicine and food that has been extensively used for thousands of years to alleviate cardiovascular diseases. It contains a significant amount of polysaccharides, yet limited research exists on whether these polysaccharides are responsible for its cardiovascular protective effects. In this study, the anti-atherosclerosis effect of the crude polysaccharides of AMB (AMBP) was evaluated using ApoE-/- mice fed a high-fat diet, along with ox-LDL-induced Thp-1 foam cells. Subsequently, guided by the inhibitory activity of foam cells formation, a major homogeneous polysaccharide named AMBP80-1a was isolated and purified, yielding 11.1 % from AMB. The molecular weight of AMBP80-1a was determined to be 10.01 kDa. AMBP80-1a was firstly characterized as an agavin-type fructan with main chains consisting of →1)-ß-d-Fruf-(2→ and →1,6)-ß-d-Fruf-(2→ linked to an internal glucose moiety, with →6)-ß-d-Fruf-(2→ and ß-d-Fruf-(2→ serving as side chains. Furthermore, the bio-activity results indicated that AMBP80-1a reduced lipid accumulation and cholesterol contents in ox-LDL-induced Thp-1 foam cell. These findings supported the role of AMBP in alleviating atherosclerosis in vivo/vitro. AMBP80-1a, as the predominant homogeneous polysaccharide in AMB, was expected to be developed as a functional agent to prevent atherosclerosis.

TAT-beclin1 treatment accelerates the development of atherosclerotic lesions in ApoE-deficient mice.

The importance of autophagy in atherosclerosis has garnered significant attention regarding the potential applications of autophagy inducers. However, the impact of TAT-Beclin1, a peptide inducer of autophagy, on the development of atherosclerotic plaques remains unclear. Single-cell omics analysis indicates a notable reduction in GAPR1 levels within fibroblasts, stromal cells, and macrophages during atherosclerosis. Tat-beclin1 (T-B), an autophagy-inducing peptide derived from Beclin1, could selectively bind to GAPR1, relieving its inhibition on Beclin1 and thereby augmenting autophagosome formation. To investigate its impact on atherosclerosic plaque progression, we established the ApoE-/- mouse model of carotid atherosclerotic plaques. Surprisingly, intravenous administration of Tat-beclin1 dramatically accelerated the development of carotid artery plaques. Immunofluorescence analysis suggested that macrophage aggregation and autophagosome formation within atherosclerotic plaques were significantly increased upon T-B treatment. However, immunofluorescence and transmission electron microscopy (TEM) analysis revealed a reduction in autophagy flux through lysosomes. In vitro, the interaction between T-B and GAPR1 was confirmed in RAW264.7 cells, resulting in the increased accumulation of p62/SQSTM1 and LC3-II in the presence of ox-LDL. Additionally, T-B treatment elevated the protein levels of p62/SQSTM1, LC3-II, and cleaved caspase 1, along with the secretion of IL-1ß in response to ox-LDL exposure. In summary, our study underscores that T-B treatment amplifies abnormal autophagy and inflammation, consequently exacerbating atherosclerotic plaque development in ApoE-/- mice.

Role of Apolipoprotein E in the Hippocampus and Its Impact following Ionizing Radiation Exposure.

Apolipoprotein E (ApoE) is a lipid carrier in both the peripheral and the central nervous systems (CNSs). Lipid-loaded ApoE lipoprotein particles bind to several cell surface receptors to support membrane homeostasis and brain injury repair. In the brain, ApoE is produced predominantly by astrocytes, but it is also abundantly expressed in most neurons of the CNS. In this study, we addressed the role of ApoE in the hippocampus in mice, focusing on its role in response to radiation injury. To this aim, 8-week-old, wild-type, and ApoE-deficient (ApoE-/-) female mice were acutely whole-body irradiated with 3 Gy of X-rays (0.89 Gy/min), then sacrificed 150 days post-irradiation. In addition, age-matching ApoE-/- females were chronically whole-body irradiated (20 mGy/d, cumulative dose of 3 Gy) for 150 days at the low dose-rate facility at the Institute of Environmental Sciences (IES), Rokkasho, Japan. To seek for ApoE-dependent modification during lineage progression from neural stem cells to neurons, we have evaluated the cellular composition of the dentate gyrus in unexposed and irradiated mice using stage-specific markers of adult neurogenesis. Our findings indicate that ApoE genetic inactivation markedly perturbs adult hippocampal neurogenesis in unexposed and irradiated mice. The effect of ApoE inactivation on the expression of a panel of miRNAs with an established role in hippocampal neurogenesis, as well as its transcriptional consequences in their target genes regulating neurogenic program, have also been analyzed. Our data show that the absence of ApoE-/- also influences synaptic functionality and integration by interfering with the regulation of mir-34a, mir-29b, and mir-128b, leading to the downregulation of synaptic markers PSD95 and synaptophysin mRNA. Finally, compared to acute irradiation, chronic exposure of ApoE null mice yields fewer consequences except for the increased microglia-mediated neuroinflammation. Exploring the function of ApoE in the hippocampus could have implications for developing therapeutic approaches to alleviate radiation-induced brain injury.

Apolipoprotein E in Alzheimer's disease trajectories and the next-generation clinical care pathway.

Alzheimer's disease (AD) is a complex, progressive primary neurodegenerative disease. Since pivotal genetic studies in 1993, the ε4 allele of the apolipoprotein E gene (APOE ε4) has remained the strongest single genome-wide associated risk variant in AD. Scientific advances in APOE biology, AD pathophysiology and ApoE-targeted therapies have brought APOE to the forefront of research, with potential translation into routine AD clinical care. This contemporary Review will merge APOE research with the emerging AD clinical care pathway and discuss APOE genetic risk as a conduit to genomic-based precision medicine in AD, including ApoE's influence in the ATX(N) biomarker framework of AD. We summarize the evidence for APOE as an important modifier of AD clinical-biological trajectories. We then illustrate the utility of APOE testing and the future of ApoE-targeted therapies in the next-generation AD clinical-diagnostic pathway. With the emergence of new AD therapies, understanding how APOE modulates AD pathophysiology will become critical for personalized AD patient care.

Activation of Nrf2 inhibits atherosclerosis in ApoE-/- mice through suppressing endothelial cell inflammation and lipid peroxidation.

BACKGROUND: Nuclear erythroid 2-related factor 2 (Nrf2), a transcription factor, is critically involved in the regulation of oxidative stress and inflammation. However, the role of endothelial Nrf2 in atherogenesis has yet to be defined. In addition, how endothelial Nrf2 is activated and whether Nrf2 can be targeted for the prevention and treatment of atherosclerosis is not explored. METHODS: RNA-sequencing and single-cell RNA sequencing analysis of mouse atherosclerotic aortas were used to identify the differentially expressed genes. In vivo endothelial cell (EC)-specific activation of Nrf2 was achieved by injecting adeno-associated viruses into ApoE-/- mice, while EC-specific knockdown of Nrf2 was generated in Cdh5CreCas9floxed-stopApoE-/- mice. RESULTS: Endothelial inflammation appeared as early as on day 3 after feeding of a high cholesterol diet (HCD) in ApoE-/- mice, as reflected by mRNA levels, immunostaining and global mRNA profiling, while the immunosignal of the end-product of lipid peroxidation (LPO), 4-hydroxynonenal (4-HNE), started to increase on day 10. TNF-α, 4-HNE, and erastin (LPO inducer), activated Nrf2 signaling in human ECs by increasing the mRNA and protein expression of Nrf2 target genes. Knockdown of endothelial Nrf2 resulted in augmented endothelial inflammation and LPO, and accelerated atherosclerosis in Cdh5CreCas9floxed-stopApoE-/- mice. By contrast, both EC-specific and pharmacological activation of Nrf2 inhibited endothelial inflammation, LPO, and atherogenesis. CONCLUSIONS: Upon HCD feeding in ApoE-/- mice, endothelial inflammation is an earliest event, followed by the appearance of LPO. EC-specific activation of Nrf2 inhibits atherosclerosis while EC-specific knockdown of Nrf2 results in the opposite effect. Pharmacological activators of endothelial Nrf2 may represent a novel therapeutic strategy for the treatment of atherosclerosis.

Identification of apolipoprotein E-derived amyloid within cholesterol granulomas of leopard geckos (Eublepharis macularius).

Apolipoprotein E (ApoE) is involved in cholesterol transport among cells and also plays an important role in amyloid formation, co-depositing with amyloid fibrils in various types of amyloidosis. Although the in vivo amyloidogenicity of ApoE has not been previously demonstrated, this study provides evidence of ApoE amyloidogenicity in leopard geckos (Eublepharis macularius), belonging to the class Reptilia. Histologically, amyloid deposits were localized within cholesterol granulomas and exhibited positive Congo red staining, with yellow to green birefringence under polarized light. On mass spectrometry-based proteomic analysis, ApoE was detected as a dominant component of amyloid; of the full length of the 274 amino acid residues, peptides derived from Leu185-Arg230 were frequently detected with non-tryptic truncations. Immunohistochemistry with anti-leopard gecko ApoE antibody showed positive reactions of amyloid deposits. These results show that ApoE is an amyloid precursor protein within the cholesterol granulomas of leopard geckos. Although further investigations are needed, the C-terminal region of ApoE involved in amyloid formation is a lipid-binding region, and there should be a relationship between amyloidogenesis and the development of cholesterol granulomas in leopard geckos. This study provides novel insights into the pathogenesis of ApoE-related diseases.