ApoE Knockout Mice Research Articles

Apolipoprotein E knockout may affect cognitive function in D-galactose-induced aging mice through the gut microbiota-brain axis.

The gut microbiota plays an important role in central nervous system (CNS) disorders. Apolipoprotein E (ApoE) can affect the composition of the gut microbiota and is closely related to the CNS. However, the mechanism by which ApoE affects cognitive dysfunction through the gut microbiota–brain axis has thus far not been investigated. In this study, we used wild-type mice and ApoE knockout (ApoE–/–) mice to replicate the aging model and examined the effects of ApoE deletion on cognitive function, hippocampal ultrastructure, synaptophysin (SYP) and postsynaptic density 95 (PSD-95) in aging mice. We also explored whether ApoE deletion affects the gut microbiota and the metabolite profile of the hippocampus in aging mice and finally examined the effect of ApoE deletion on lipids and oxidative stress in aging mice. The results showed that the deletion of ApoE aggravated cognitive dysfunction, hippocampal synaptic ultrastructural damage and dysregulation of SYP and PSD-95 expression in aging mice. Furthermore, ApoE deletion reduced gut microbial makeup in aging mice. Further studies showed that ApoE deletion altered the hippocampal metabolic profile and aggravated dyslipidemia and oxidative stress in aging mice. In brief, our findings suggest that loss of ApoE alters the composition of the gut microbiota, which in turn may affect cognitive function in aging mice through the gut microbiota–brain axis.

UFMylation Is Activated in Atherosclerosis of ApoE Knockout Mice.

UFMylation is a novel ubiquitin-like system that deals with complex and fine-tuned cellular activities and is closely related to endoplasmic reticulum stress. Our previous study indicated that UFMylation is activated in vascular remodeling models. However, the role of UFMylation in atherosclerosis (AS) is unclear. In this study, we investigated changes in UFMylation in ApoE knockout (ApoE-KO) mice. We found that UFMylation was significantly activated in ApoE-KO mice fed a high-fat diet for 46 weeks. Consistently we observed that vascular smooth muscle cells (VSMCs) treated with oxidized low-density lipoprotein (oxLDL) showed UFMylation activation in a time-dependent manner. UFM1-overexpressing mice were generated using transgenic (Tg) technique and bred with ApoE-KO mice to generate ApoE-KO/UFM1-Tg mice. We found that the degree of AS did not vary compared with that of the control. Similarly, overexpression of active UFM1 failed to alter oxLDL-induced proliferation of VSMCs. These findings indicate that UFMylation is activated in AS, but overexpression of UFM1 does not alter the development of AS in ApoE-KO mice.

Metabolic Abnormalities Linked to Auditory Pathways in ApoE-Knockout HEI-OC1 Cells: A Transcription-Metabolism Co-Analysis.

Lipid transporter protein apolipoprotein E (APOE) has contributed to functional studies of various organ functions. Animals with ApoE knockout (KO) have been used to study atherosclerosis and hyperlipidemia while an increasing number of researchers have recently focused on the association of ApoE with hearing loss. A study found that ApoE KO mice experience sensorineural hearing loss and hair cell loss, but the exact mechanism is unclear. To explore the potential relationship between ApoE and hearing loss, we used HEI-OC1 cells (House Ear Institute-Organ of Corti) with Corti apparatus properties to reveal cell changes after ApoE knockout by combined transcriptome and metabolomic analysis. We found that glutamate deficiency, caused by reduced expression of glutamine transporter proteins, was a key correlate of basal metabolism and that inadequate glutamate causes apoptosis by reducing the cells’ resistance to external damage. Our study provides a reference mechanism for hearing loss due to ApoE KO.

Trypanosoma cruzi infection increases atherosclerotic lesion in ApoE-deficient mice

Apolipoprotein E (ApoE) is the major ligand for the transporting and removal of chylomicrons and lipoproteins by the liver. Since the creation of the ApoE-knockout mice, it is well established that ApoE deficiency results in spontaneous atherosclerosis in aged animals. Atherosclerosis is also observed in animals infected with Trypanosoma cruzi, a protozoan that elicits a systemic inflammatory response in mammalian hosts, culminating in damage to cardiac, neuronal, and endothelial cells. Pro-atherogenic effects related to the experimental infection with T. cruzi may be induced by inflammatory components affecting the vascular wall. Herein, we evaluated whether infection with different strains of T. cruzi worsened the atherogenic lesions observed in aged ApoE−/− mice. After four weeks of infection with Berenice-78 (Be-78) or Colombian (Col) strains of the parasite, mice presented increased CCL2 and CCL5 production and high migration of inflammatory cells to cardiac tissue. Although the infection with either strain did not affect the survival rate, only the infection with Col strain caused abundant parasite growth in blood and heart and increased aortic root lesions in ApoE−/− mice. Our findings show, for the first time that ApoE exerts a protective anti-atherosclerotic role in the aortic root of mice in the acute phase of experimental infection with the Col strain of T. cruzi. Further studies should target ApoE and nutritional interventions to modulate susceptibility to cardiovascular disabilities after T. cruzi infection, minimizing the risk of death in both experimental animals and humans.

Adenine-Induced Nephropathy Reduces Atherosclerosis in ApoE Knockout Mice.

Background: Cardiovascular events are the main cause of death in patients with chronic kidney disease. We hypothesize that the protective effects of renal cholesterol and vitamin D3 metabolism are lost under this condition. Nephropathy was induced by adenine in Apolipoprotein E knockout mice. The atherosclerotic phenotype was compared to mice with normal renal function. Methods: Mice were fed a western diet ±0.15% adenine. Urine and feces were collected to assess renal function and fecal output. Atherosclerosis, serum lipoprotein composition and functionality, hepatic lipids, and expression of genes involved in lipid metabolism, vitamin D3 and Na+ homeostasis, were assessed. Bones were analyzed by microCT. Results: Mice fed with adenine showed enhanced urinary Na+, Ca2+, and Pi excretion, reduced urinary pH, UreaUrine/UreaSerum, and CreatinineUrine/CreatinineSerum ratios. They developed less atherosclerosis. Lipoproteins in serum and hepatic lipids remained unchanged. Cholesterol efflux increased. Fecal output of cholesteryl ester and triglycerides increased. In the liver, mRNA levels of Cyp27a1, Cyp7a1, and Scarb1 increased; in the kidneys, Slc9a3, Slc12a3, Vdr, and Cyp24a1 decreased. Adenine increased cholesterol efflux in vitro. Tibias were shorter. Conclusion: Adenine induced tubular damage and was athero-protective because of enhanced cholesterol efflux and lipids elimination in feces. Bone growth was also affected.

Broad-acting therapeutic effects of miR-29b-chitosan on hypertension and diabetic complications

MicroRNA miR-29 promotes endothelial function in human arterioles in part by targeting LYPLA1 and increasing nitric oxide production. In addition, miR-29 is a master inhibitor of extracellular matrix gene expression, which may attenuate fibrosis but could also weaken tissue structure. The goal of this study was to test whether miR-29 could be developed as an effective, broad-acting, and safe therapeutic. Substantial accumulation of miR-29b and effective knockdown of Lypla1 in several mouse tissues were achieved using a chitosan-packaged, chemically modified miR-29b mimic (miR-29b-CH-NP) injected systemically at 200μg/kg body weight. miR-29b-CH-NP, injected once every 3days, significantly attenuated angiotensin II-induced hypertension. In db/db mice, miR-29b-CH-NP treatment for 12weeks decreased cardiac and renal fibrosis and urinary albuminuria. In uninephrectomized db/db mice, miR-29b-CH-NP treatment for 20weeks significantly improved myocardial performance index and attenuated proteinuria. miR-29b-CH-NP did not worsen abdominal aortic aneurysm in ApoE knockout mice treated with angiotensin II. miR-29b-CH-NP caused aortic root fibrotic cap thinning in ApoE knockout mice fed a high-cholesterol and high-fat diet but did not worsen the necrotic zone or mortality. In conclusion, systemic delivery of low-dose miR-29b-CH-NP is an effective therapeutic for several forms of cardiovascular and renal disease in mice.

Dietary choline increases plasma concentration of gut-derived uremic toxins and metabolites associated with chronic kidney disease progression in apoE-knockout mice with elevated HDL

Background and Aims : Experimental and observational studies have highlighted a positive correlation between increased plasma concentration of choline-derived trimethylamine-N-oxide (TMAO) and adverse cardiovascular events. This study was aimed at investigating how the plasma metabolome of mice prone to atherosclerosis development was modulated by HDL levels and the dietary intake of choline.

Changes in the liver proteome in apoE knockout mice exposed to inhalation of silica nanoparticles indicate mitochondrial damage and impairment of ER stress responses associated with microvesicular steatosis

The adverse effects of air pollution on the cardiovascular system have been well documented. Nonalcoholic fatty liver disease (NAFLD) is an independent risk factor for cardiovascular events. However, the influence of exposure to airborne particles on the development of NAFLD is less recognised. The aim of this study was to investigate the impact of silica nanoparticles (SiNPs) on the development of liver steatosis. We used molecular and proteomic SWATH-MS methods to investigate the changes in the liver proteome of apolipoprotein E-knockout mice (apoE−/− mice) exposed to SiNPs for 4 months in a whole-body exposure chamber. Exposure to SiNPs evoked microvesicular liver steatosis in apoE−/− mice. Quantitative liver proteomics showed significant downregulation of ribosomal proteins and endoplasmic reticulum proteins. Gene expression analysis revealed a reduced level of proteins related to endoplasmic reticulum stress. Treatment with SiNPs decreased mitochondrial membrane potential and increased the production of reactive oxygen species in cultured HepG2 cells. This is the first report that inhalation exposure to SiNPs induces microvesicular steatosis and significant changes in the liver proteome in vivo. Our results highlight the important role of silica and point to the ER stress response and mitochondrial dysfunction as potential mechanisms responsible for the increase in fatty liver by SiNPs.

Neuronal ApoE Regulates the Cell-to-Cell Transmission of α-Synuclein.

The presence of protein inclusions, called Lewy bodies (LBs) and Lewy neurites (LNs), in the brain is the main feature of Parkinson’s disease (PD). Recent evidence that the prion-like propagation of α-synuclein (α-syn), as a major component of LBs and LNs, plays an important role in the progression of PD has gained much attention, although the molecular mechanism remains unclear. In this study, we evaluated whether neuronal ApoE regulates the cell-to-cell transmission of α-syn and explored its molecular mechanism using in vitro and in vivo model systems. We demonstrate that neuronal ApoE deficiency attenuates both α-syn uptake and release by downregulating LRP-1 and LDLR expression and enhancing chaperone-mediated autophagy activity, respectively, thereby contributing to α-syn propagation. In addition, we observed that α-syn propagation was attenuated in ApoE knockout mice injected with pre-formed mouse α-syn fibrils. This study will help our understanding of the molecular mechanisms underlying α-syn propagation.

Platelet-derived extracellular vesicles encapsulate microRNA-34c-5p to ameliorate inflammatory response of coronary artery endothelial cells via PODXL-mediated P38 MAPK signaling pathway

Background and aimsLow-grade chronic inflammation was reported to serve as a distinctive pathophysiologic feature of coronary artery disease (CAD), the leading cause of death around the world. Herein, the current study aimed to explore whether and how microRNA-34c-5p (miR-34c-5p), a miRNA enriched in extracellular vesicles (EVs) originated from the activated platelet (PLT-EVs), affects the inflammation of human coronary artery endothelial cells (HCAECs). Methods and resultsHCAECs were established as an in vitro cell model using oxidized low-density lipoprotein (ox-LDL). miR-34c-5p, an abundant miRNA in PLT-EVs, can be transferred to HCAECs and target PODXL by binding to its 3′UTR. Gain- and loss-of-function experiments of miR-34c-5p and podocalyxin (PODXL) were performed in ox-LDL-induced HCAECs. Subsequently, HCAECs were subjected to co-culture with PLT-EVs, followed by detection of the expression patterns of key pro-inflammatory factors. Either miR-34c-5p mimic or PLT-EVs harboring miR-34c-5p attenuated the ox-LDL-evoked inflammation in HCAECs by suppressing interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α). By blocking the P38 MAPK signaling pathway, miR-34c-5p-mediated depletion of PODXL contributed to protection against ox-LDL-induced inflammation. In vitro findings were further validated by findings observed in ApoE knock-out mice. Additionally, miR-34c-5p in PLT-EVs showed an athero-protective role in the murine model. ConclusionAltogether, our findings highlighted that miR-34c-5p in PLT-EVs could alleviate inflammation response in HCAECs by targeting PODXL and inactivation of the P38 MAPK signaling pathway.

Exercise training inhibits atherosclerosis progression and reduces VE-cadherin levels within atherosclerotic plaques in hypercholesterolemic mice

Vascular endothelial-cadherin (VE-cadherin), matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) have emerged as key-factors of atherogenesis. The aim of this study was to evaluate the effects of exercise training (ET) on those key-factors in relation to the progression of atherosclerotic lesions in hypercholesterolemic mice. Thirty male, apoE knockout (apoE−/−) mice were randomly assigned to the following equivalent groups: 1) CO-control: High-fat diet (HFD) administration for 12 weeks. 2) EX-exercise: HFD administration as in CO, and during the last 4 weeks (9th −12th week) ET on treadmill (5sessions/week, 60min/session). At the end of study, blood samples were obtained and all mice were sacrificed. Aortic roots were excised and analysed regarding the percentage of aortic stenosis, and the relative concentrations of collagen, elastin, VE-cadherin, MMP-8,-9 and TIMP-1,-2 within the atherosclerotic lesions. Aortic stenosis was significantly lower in the EX than the CO group (39.63 ± 7.22% vs 62.04 ± 8.55%; p < 0.001), along with considerable increase in fibrous cap thickness and of collagen and elastin contents within plaques (p < 0.05). Compared to controls, exercised-treated mice showed reduced intra-plaque relative concentrations of VE-cadherin (15.09 ± 1.89% vs 23.49 ± 3.01%, p < 0.001), MMP-8 (8.51 ± 2.24% vs 18.51 ± 4.08%, p < 0.001) and MMP-9 (12.1 ± 4.86% vs 18.88 ± 6.23%, p < 0.001). Inversely, the relative concentrations of TIMP-1 and TIMP-2 in the ET group were considerably higher by 62.5% and 31.2% than in the EX group (p < 0.05), respectively. Finally, body weight and lipids concentrations did not differ between groups at the end of the study (p > 0.05). ET treatment induced regression of established atherosclerotic lesions in apoE−/− mice and improved their stability. Those effects seemed to be mediated by favourable modification of VE-cadherin, MMPs and TIMPs.

Loss of RAGE prevents chronic intermittent hypoxia-induced nonalcoholic fatty liver disease via blockade of NF-кB pathway.

In recent years, receptor for advanced glycation end-products (RAGE) has been documented to induce liver fibrosis and inflammatory reaction. Further, microarray data analysis of this study predicted high expression of RAGE in non-alcoholic fatty liver disease (NAFLD). However, its specific mechanisms remain to be elucidated. Hence, this study is aimed at investigating the mechanistic insights of RAGE in chronic intermittent hypoxia (CIH)-induced NAFLD. ApoE knockout (ApoE-/-) mice were exposed to CIH to induce NAFLD, and primary hepatocytes were also exposed to CIH to mimic in vitro setting. Accordingly, we found that RAGE and NF-κB were upregulated in the liver tissues of CIH-induced NAFLD mice and CIH-exposed hepatocytes. Depleted RAGE attenuated CIH-induced hepatocyte injury, lipid deposition, and inflammation. The relationship between RAGE and NF-κB was analyzed by in silico analysis and correlation analysis. It was demonstrated that knockdown of RAGE inhibited the NF-кB pathway, thus alleviating CIH-induced disorders in hepatocytes. Moreover, in vivo experiments also verified that depletion of RAGE alleviated CIH-induced NAFLD by inhibiting NF-кB pathway. Collectively, loss of RAGE blocked the NF-кB pathway to alleviate CIH-induced NAFLD, therefore, highlighting a potential hepatoprotective target for treating NAFLD.

Atherosclerosis Plaque Reduction by Lycopene Is Mediated by Increased Energy Expenditure through AMPK and PPARα in ApoE KO Mice Fed with a High Fat Diet.

Lycopene is a carotenoid found in tomatoes that has potent antioxidant activity. The Mediterranean diet is particularly rich in lycopene, which has well-known beneficial effects on cardiovascular health. We tested the effects of lycopene extract in a group of 20 ApoE knockout mice, fed with a high fat western diet for 14 weeks. Starting from week 3 and up to week 14, the mice were randomly divided into two groups that received lycopene (n = 10) by oral suspension every day at the human equivalent dose of 60 mg/day (0.246 mg/mouse/day), or the vehicle solution (n = 10). The lycopene administration reduced triglycerides and cholesterol blood levels starting from week 6 and continuing through to the end of the experiment (p < 0.001). This reduction was mediated by an enhanced liver expression of PPAR-α and AMPK-α and reduced SREBP levels (p < 0.0001). As a histological red-out, the extent of atherosclerotic plaques and the intima–media thickness in the aorta were significantly reduced by lycopene. In this context, lycopene augmented the Nrf-2 positivity staining in the endothelium, thereby confirming that its antioxidant activity was mediated by this nuclear factor. The positive results obtained in this pre-clinical model further support the use of lycopene extracts to reduce atherosclerosis.

Effects of toxic apolipoprotein E fragments on Tau phosphorylation and cognitive impairment in neonatal mice under sevoflurane anesthesia.

BackgroundAnesthesia induces Tau phosphorylation and cognitive impairment in young, but not adult, mice. Apolipoprotein E (ApoE) may play a protective role in neuronal activity and injury repair, whereas its toxic fragments are reported to induce neurodegeneration and neurocognitive impairment in patients with Alzheimer's disease (AD). Therefore, we set out to test the hypothesis that the difference in ApoE fragments, but not the full‐length ApoE, contributes to the difference in Tau phosphorylation and neurocognitive functions following sevoflurane anesthesia in young mice.MethodsSevoflurane was administered to wild‐type (WT), ApoE‐knockout (ApoE‐KO), ApoE3‐targeted replacement (ApoE3 expresses both full‐length and fragmented ApoE), and ApoE2‐targeted replacement (ApoE2 only expresses full‐length ApoE) mice. The mRNA and protein levels of ApoE, phosphorylated Tau (pTau), and cognitive function were tested in the mice.ResultsSevoflurane anesthesia enhanced ApoE mRNA, total ApoE, full‐length ApoE, ApoE fragments, Tau phosphorylation (AT8 and PHF1), and cognitive impairment in young mice, but not in adult mice. ApoE2, but not ApoE3 or ApoE‐KO, mice showed reduced sevoflurane‐induced pTau elevation and cognitive impairment.ConclusionThese data suggest that elevated ApoE fragments rather than full‐length ApoE might be one of the underlying mechanisms of age‐dependent Tau phosphorylation and cognitive impairment in young mice following sevoflurane anesthesia.

Bone disturbances and progression of atherosclerosis in ApoE knockout mice.

Epidemiological evidence supports a link between atherosclerosis and osteoporosis. These conditions might share common pathophysiological mechanisms, with inflammation being one of the hypotheses.Apolipoprotein E deficient mice (ApoE-/-) develop atherosclerotic lesions spontaneously, further aggravated by a high-fat diet. Their bone remodelling is also disturbed. We hypothesised that a proinflammatory state could be a common contributive factor for vessel and bone disturbances observed in this animal model. We evaluated vessels and bones of ApoE-/- and control C57BL/6 (B6) female mice fed a high-fat diet in five time-points (8, 16, 20, 24 and 28 weeks of age) and quantified the development of atherosclerotic lesions, analysed gene expression of inflammatory and bone remodelling proteins (IL-1β, IL-6, IL-17A, TNF, RANKL, and OPG), measured serum bone turnover markers (P1NP and CTX-I), performed bone (L3-L4 vertebras) histomorphometric analysis and evaluated biomechanical properties of bones. We compared the outcomes of B6 and ApoE-/- groups at each time-point and, within each group, over time. Atherosclerotic lesions developed as previously described for ApoE-/- mice, but no significant differences were found in bone histomorphometry or biomechanical properties between ApoE-/- and B6 mice. Also, gene expression (either in bones or aortas) and serum biomarkers were similar in both groups. When considering over time evaluations we found that bone histomorphometry changes were similar between ApoE-/- and B6 mice, but CTX-I/P1NP ratio was significantly increased (meaning higher resorption than bone formation) in ApoE-/- as compared to B6 mice. Our study suggests that inflammation is not the principal driver for atherosclerosis progression and bone disturbances in this animal model.

Apolipoprotein E ε4 Mediates Myelin Breakdown by Targeting Oligodendrocytes in Sporadic Alzheimer Disease.

White matter degradation in the frontal lobe is one of the earliest detectable changes in aging and Alzheimer disease. The ε4 allele of apolipoprotein E (APOE4) is strongly associated with such myelin pathology but the underlying cellular mechanisms remain obscure. We hypothesized that, as a lipid transporter, APOE4 directly triggers pathology in the cholesterol-rich myelin sheath independent of AD pathology. To test this, we performed immunohistochemistry on brain tissues from healthy controls, sporadic, and familial Alzheimer disease subjects. While myelin basic protein expression was largely unchanged, in frontal cortex the number of oligodendrocytes (OLs) was significantly reduced in APOE4 brains independent of their Braak stage or NIA-RI criteria. This high vulnerability of OLs was confirmed in humanized APOE3 or APOE4 transgenic mice. A gradual decline of OL numbers was found in the aging brain without associated neuronal loss. Importantly, the application of lipidated human APOE4, but not APOE3, proteins significantly reduced the formation of myelinating OL in primary cell culture derived from Apoe-knockout mice, especially in cholesterol-depleted conditions. Our findings suggest that the disruption of myelination in APOE4 carriers may represent a direct OL pathology, rather than an indirect consequence of amyloid plaque formation or neuronal loss.

Abstract 4152: Development of an immune modulating and tumor inhibiting hyaluronic acid nanoparticle encapsulated with Avasimibe for the treatment of cancer patients with comorbid atherosclerosis

Abstract Cancer and cardiovascular diseases are the leading causes of death globally. Given the high percentage of cancer patients with co-existing atherosclerosis due to many shared risk factors, the development of novel cancer therapeutic agents with strong anti-tumor efficacy and therapeutic benefit on atherosclerosis can significantly improve the outcome of cancer therapy. Immune checkpoint inhibition (ICI) therapy using therapeutic antibodies has shown promises in the treatment of several types of human cancers. However, many cancer patients showed a poor response due to low delivery efficiency, lack of effector T cells, an immunosuppressive tumor microenvironment, and intrinsic resistance in solid tumors. Increasing numbers of patients developed immunotherapy related adverse effects (irAEs). It is well known that macrophages and effector T cells drive the progression of atherosclerosis. Recent clinical studies revealed that cancer patients received ICI therapy have 3-fold higher risk of atherosclerosis and 3-fold increases in plaque progression detected by PET imaging. To enhance therapeutic efficacy of tumor immunotherapy and decrease irAEs, we have developed a hyaluronic acid nanoparticle (HANP) conjugated with PD1 mimetic peptides that target and block PD-L1 function, and encapsulated with Avasimibe (PD1Y-HANP/Ava). Avasimibe is a multifunctional agent that decreases cholesterol accumulation, inhibits tumor growth, and enhances immune response by activating cytotoxic T cells. We found that systemic administrations of PD1Y-HANP/Ava led to targeted delivery into tumors and atherosclerotic plaques in a dual colon cancer and atherosclerosis mouse model, established by injecting mouse colon tumor cells into Apoe knockout mice on a high fat diet. PD1Y-HANP/Ava treatments resulted in 78% of tumor growth inhibition. Following surgical resection of residual tumors, 80% of PD1Y-HANP/Ava treated mice had disease-free survival of &amp;gt;120 days and were protected from tumor growth after tumor cell re-challenging. Histological analysis of the major arteries revealed that the volume of atherosclerotic plaques decreased 70% in the mice treated with PD1Y-HANP/Ava compared the no-treated mice. Our results showed that PD1Y-HANP/Ava treatment increased infiltration of CD8+ T effector and dendritic cells, and activated cytotoxic T cells in mouse colon tumors. Colon tumor specific antibodies were detected in the mouse serum following PD1Y-HANP/Ava treatment. However, the levels of CD8+ T cells, dendritic cells, and macrophages were decreased in the atherosclerotic plaques, which could prevent ICI-induced cardiovascular irAEs. Results of this study should provide us with preclinical evidence for translational development of targeted immunotherapy for colon cancer patients with comorbid atherosclerosis. Citation Format: Lei Zhu, Weiping Qian, Minglong Chen, Tongrui Liu, Charles A. Staley, Bassel El-Rayes, Hanjoong Jo, Lily Yang. Development of an immune modulating and tumor inhibiting hyaluronic acid nanoparticle encapsulated with Avasimibe for the treatment of cancer patients with comorbid atherosclerosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4152.

Indoleamine 2,3-Dioxygenase 1 Deletion-Mediated Kynurenine Insufficiency in Vascular Smooth Muscle Cells Exacerbates Arterial Calcification.

IDO1 (indoleamine 2,3-dioxygenase 1) is the rate-limiting enzyme for tryptophan metabolism. IDO1 malfunction is involved in the pathogenesis of atherosclerosis. Vascular smooth muscle cells (VSMCs) with an osteogenic phenotype promote calcification and features of plaque instability. However, it remains unclear whether aberrant IDO1-regulated tryptophan metabolism causes VSMCs osteogenic reprogramming and calcification. We generated global Apoe (apolipoprotein E) and Ido1 double knockout mice, and Apoe knockout mice with specific deletion of IDO1 in VSMCs or macrophages. Arterial intimal calcification was evaluated by a Western diet-induced atherosclerotic calcification model. Global deficiency of IDO1 boosted calcific lesion formation without sex bias in vivo. Conditional IDO1 loss of function in VSMCs rather than macrophages promoted calcific lesion development and the abundance of RUNX2 (runt-related transcription factor 2). In contrast, administration of kynurenine via intraperitoneal injection markedly delayed the progression of intimal calcification in parallel with decreased RUNX2 expression in both Apoe-/- and Apoe-/-Ido1-/- mice. We found that IDO1 deletion restrained RUNX2 from proteasomal degradation, which resulted in enhanced osteogenic reprogramming of VSMCs. Kynurenine administration downregulated RUNX2 in an aryl hydrocarbon receptor-dependent manner. Kynurenine acted as the endogenous ligand of aryl hydrocarbon receptor, controlled resultant interactions between cullin 4B and aryl hydrocarbon receptor to form an E3 ubiquitin ligase that bound with RUNX2, and subsequently promoted ubiquitin-mediated instability of RUNX2 in VSMCs. Serum samples from patients with coronary artery calcification had impaired IDO1 activity and decreased kynurenine catabolites compared with those without calcification. Kynurenine, an IDO1-mediated tryptophan metabolism main product, promotes RUNX2 ubiquitination and subsequently leads to its proteasomal degradation via an aryl hydrocarbon receptor-dependent nongenomic pathway. Insufficient kynurenine exerts the deleterious role of IDO1 ablation in promoting RUNX2-mediated VSMCs osteogenic reprogramming and calcification in vivo.

Arsenic Exposure, Blood DNA Methylation, and Cardiovascular Disease.

Epigenetic dysregulation has been proposed as a key mechanism for arsenic-related cardiovascular disease (CVD). We evaluated differentially methylated positions (DMPs) as potential mediators on the association between arsenic and CVD. Blood DNA methylation was measured in 2321 participants (mean age 56.2, 58.6% women) of the Strong Heart Study, a prospective cohort of American Indians. Urinary arsenic species were measured using high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry. We identified DMPs that are potential mediators between arsenic and CVD. In a cross-species analysis, we compared those DMPs with differential liver DNA methylation following early-life arsenic exposure in the apoE knockout (apoE-/-) mouse model of atherosclerosis. A total of 20 and 13 DMPs were potential mediators for CVD incidence and mortality, respectively, several of them annotated to genes related to diabetes. Eleven of these DMPs were similarly associated with incident CVD in 3 diverse prospective cohorts (Framingham Heart Study, Women's Health Initiative, and Multi-Ethnic Study of Atherosclerosis). In the mouse model, differentially methylated regions in 20 of those genes and DMPs in 10 genes were associated with arsenic. Differential DNA methylation might be part of the biological link between arsenic and CVD. The gene functions suggest that diabetes might represent a relevant mechanism for arsenic-related cardiovascular risk in populations with a high burden of diabetes.

Dietary Ellagic Acid Ameliorates Functionality of Reverse Cholesterol Transport in apoE-Deficient Mice

ObjectivesHigh levels of plasma LDL cholesterol are an important determinant of atherosclerotic lesion formation. The disruption of the process of cholesterol efflux or reverse cholesterol transport (RCT) of peripheral cells may promote atherogenesis. The aim of the current study was to investigate whether ellagic acid alleviated atherosclerotic development through activating RCT pathway in apoE knockout (KO) mice. MethodsWild type mice and apoE KO mice were fed high-fat high-cholesterol Paigen’s diets for 10 weeks to induce hypercholesterolemia and atherosclerosis, and concomitantly received 10 mg/kg ellagic acid via gavage. ResultsSupplying ellagic acid to apoE KO mice reduced the number of eosinophils and basophils. Ellagic acid enhanced induction of apoE and ABCG1 in oxidized LDL-exposed macrophages responsible for cholesterol efflux associated with RCT. Oral administration of ellagic acid displayed lipid-lowering effect with a reduction of atherogenic index highly elevated by Paigen’s diets in apoE KO mice. Plasma levels of cholesterol ester transport protein and phospholipid transport protein involved in RCT were elevated in mice lack of apoE gene, which was substantially reduced by supplementing ellagic acid to Paigen’s diet-fed mice. In addition, ellagic acid attenuated hepatic lipid accumulation from circulation via RCT process, evidenced by staining of H&E and Oil red O in apoE KO mice. Furthermore, the supplementation of 10 mg/kg ellagic acid favorably influenced the transcriptional levels of hepatic lipoprotein receptor of LDL receptor and scavenger receptor-B1 in Paigen’s diet-fed apoE KO mice. Finally, the signaling of PPAR-liver X receptor α appeared to be involved in the lipid-lowering ability of ellagic acid in apoE KO mice. ConclusionsEllagic acid may be an athero-protective dietary compound encumbering diet-induced atherogenesis though improving the RCT functionality. Funding SourcesThis work was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2021R1A6A1A03044501).