Male ApoE Research Articles

Weight trajectories in aging humanized APOE mice with translational validity to human Alzheimer's risk population: A retrospective analysis.

Translational validity of mouse models of Alzheimer's disease (AD) is variable. Because change in weight is a well-documented precursor of AD, we investigated whether diversity of human AD risk weight phenotypes was evident in a longitudinally characterized cohort of 1,196 female and male humanized APOE (hAPOE) mice, monitored up to 28 months of age which is equivalent to 81 human years. Autoregressive Hidden Markov Model (AHMM) incorporating age, sex, and APOE genotype was employed to identify emergent weight trajectories and phenotypes. In the hAPOE-AD mouse cohort, five distinct weight trajectories emerged: three trajectories were associated with a weight loss phenotype (36% of mice, n = 426), one with weight gain (13% of mice, n = 152), and one trajectory of no change in weight (34% of mice, n = 403). The AHMM model findings were validated with post-hoc survival analyses, revealing differences in survival rates across the five identified phenotypes. Further validation was performed using body composition and plasma β-amyloid data from mice within the identified gain, loss and stable weight trajectories. Weight gain trajectory was associated with elevated plasma β-amyloid levels, higher body fat composition, lower survival rates and a greater proportion of APOE4/4 carriers. In contrast, weight loss was associated with greater proportion of hAPOE3/4 carriers, better survival rates and was predominantly male. The association between weight change and AD risk observed in humans was mirrored in the hAPOE-AD mouse model. Weight trajectories of APOE3/3 mice were equally distributed across weight gain, loss and stability. Surprisingly, despite genetic uniformity, comparable housing, diet and handling, distinct weight trajectories and divergence points emerged for subpopulations. These data are consistent with the heterogeneity observed in the human population for change in body weight during aging and highlight the importance of longitudinal phenotypic characterization of mouse aging to advance the translational validity of preclinical AD mouse models.

Astragali Radix-Angelicae Sinensis Radix inhibits the activation of vascular adventitial fibroblasts and vascular intimal proliferation by regulating the TGF-β1/Smad2/3 pathway.

Astragali Radix-Angelicae Sinensis Radix is an important traditional Chinese medicine used for the treatment of cardiovascular diseases. Our previous studies have shown that Astragali Radix-Angelicae Sinensis Radix can inhibit vascular intimal hyperplasia and improve the blood vessel wall's ECM deposition, among which six main active components can be absorbed into the blood, suggesting that these components may be the main pharmacodynamic substances of Astragali Radix-Angelicae Sinensis Radix against vascular intimal hyperplasia. A mouse model of atherosclerosis was used to study the relationship between the anti-intimal hyperplasia effect of Astragali Radix-Angelicae Sinensis Radix and the inhibition of VAF activation and ECM synthesis. Furthermore, an in vitro rat VAF activation model was used. The effects of the main active ingredients of Astragali Radix-Angelicae Sinensis Radix on the proliferation, migration and ECM synthesis of VAF were observed. The mechanism of its action was investigated by focusing on TGF-β1/Smads signaling pathway. Male ApoE-/- mice were used to establish an AS model. Observe the morphological changes of blood vessels, the expression of Vimentin, α-SMA, ECM-related factors and TGF-β1/Smads signaling pathway-related proteins. Ang Ⅱ was used to induce the VAF activation model. The cell activity, cell proliferation, cell migration, cell phenotypic markers, ECM-related factors, cell cycle regulation-related proteins and TGF-β1/Smads signaling pathway-related proteins were determined. On this basis, TGF-β1/Smads signaling pathway agonists and inhibitors were used to study the effects of the compatibility of six active components on TGF-β1/Smads signaling pathway. Astragali Radix-Angelicae Sinensis Radix can reduce aortic intimal hyperplasia, inhibit the expression of aortic α-SMA, Vimentin, ECM components, TGF-β1, p-Samd2 and p-Samd3. Cell experiments showed that the six active ingredients could inhibit the proliferation and migration of VAF to varying degrees, inhibit the expression of α-SMA, cell cycle promoters, ECM components, up-regulate the expression of Vimentin, P21, MMP2 and MMP9. The above effects were enhanced after the combination of the six components. The 6 components and their combinations could inhibit the expression of TGF-β1/Smads signaling pathway-related proteins and up-regulate the expression of Samd7 to varying degrees. The above effects were enhanced after the combination of the 6 components. TGF-β1/Smads signaling pathway inhibitor LY2157299 showed similar effects with the six components. The inhibitory effects of the six active ingredients on TGF-β1/Smads signaling pathway-related proteins and the promotion of Smad7 expression were attenuated when agonists were added into the six active ingredient combinations. However, adding TGFβ1/Smads signaling pathway inhibitor EGF to the six active ingredient combinations had no effect on the above effects. Astragali Radix-Angelicae Sinensis Radix can inhibit intimal hyperplasia, VAF activation, and ECM synthesis in atherosclerosis. The six active ingredients may be the main pharmacological substances of Astragali Radix-Angelicae Sinensis Radix to inhibit the activation of VAF, and the combination of six ingredients can enhance their effects, which may be mediated by inhibiting the activation of the TGF-β1/Smad2/3 signaling pathway.

Single-Cell Multi-Omics Profiling of Immune Cells Isolated from Atherosclerotic Plaques in Male ApoE Knockout Mice Exposed to Arsenic.

Millions worldwide are exposed to elevated levels of arsenic that significantly increase their risk of developing atherosclerosis, a pathology primarily driven by immune cells. While the impact of arsenic on immune cell populations in atherosclerotic plaques has been broadly characterized, cellular heterogeneity is a substantial barrier to in-depth examinations of the cellular dynamics for varying immune cell populations. This study aimed to conduct single-cell multi-omics profiling of atherosclerotic plaques in apolipoprotein E knockout (ApoE-/-) mice to elucidate transcriptomic and epigenetic changes in immune cells induced by arsenic exposure. The ApoE-/- mice were fed a high-fat diet and were exposed to either arsenic in drinking water or a tap water control, and single-cell multi-omics profiling was performed on atherosclerotic plaque-resident immune cells. Transcriptomic and epigenetic changes in immune cells were analyzed within the same cell to understand the effects of arsenic exposure. Our data revealed that the transcriptional profile of macrophages from arsenic-exposed mice were significantly different from that of control mice and that differences were subtype specific and associated with cell-cell interaction and cell fates. Additionally, our data suggest that differences in arsenic-mediated changes in chromosome accessibility in arsenic-exposed mice were statistically more likely to be due to factors other than random variation compared to their effects on the transcriptome, revealing markers of arsenic exposure and potential targets for intervention. These findings in mice provide insights into how arsenic exposure impacts immune cell types in atherosclerosis, highlighting the importance of considering cellular heterogeneity in studying such effects. The identification of subtype-specific differences and potential intervention targets underscores the significance of understanding the molecular mechanisms underlying arsenic-induced atherosclerosis. Further research is warranted to validate these findings and explore therapeutic interventions targeting immune cell dysfunction in arsenic-exposed individuals. https://doi.org/10.1289/EHP14285.

PAC1 Agonist Maxadilan Reduces Atherosclerotic Lesions in Hypercholesterolemic ApoE-Deficient Mice.

A possible involvement of immune- and vasoregulatory PACAP signaling at the PAC1 receptor in atherogenesis and plaque-associated vascular inflammation has been suggested. Therefore, we tested the PAC1 receptor agonist Maxadilan and the PAC1 selective antagonist M65 on plaque development and lumen stenosis in the ApoE-/- atherosclerosis model for possible effects on atherogenesis. Adult male ApoE-/- mice were fed a cholesterol-enriched diet (CED) or standard chow (SC) treated with Maxadilan, M65 or Sham. Effects of treatment on atherosclerotic plaques, lumen stenosis, apoptosis and pro-inflammatory signatures were analyzed in the brachiocephalic trunk (BT). The percentage of Maxadilan treated mice exhibiting plaques under SC and CED was lower than that of Sham or M65 treatment indicating opposite effects of Maxadilan and M65. Maxadilan application inhibited lumen stenosis in SC and CED mice compared to the Sham mice. In spite of increased cholesterol levels, lumen stenosis of Maxadilan-treated mice was similar under CED and SC. In contrast, M65 under SC or CED did not reveal a significant influence on lumen stenosis. Maxadilan significantly reduced the TNF-α-immunoreactive (TNF-α+) area in the plaques under CED, but not under SC. In contrast, the IL-1β+ area was reduced after Maxadilan treatment in SC mice but remained unchanged in CED mice compared to Sham mice. Maxadilan reduced caspase-3 immunoreactive (caspase-3+) in the tunica media under both, SC and CED without affecting lipid content in plaques. Despite persistent hypercholesterolemia, Maxadilan reduces lumen stenosis, apoptosis and TNF-α driven inflammation. Our data suggest that Maxadilan provides atheroprotection by acting downstream of hypercholesterolemia-induced vascular inflammation. This implicates the potential of PAC1-specific agonist drugs against atherosclerosis even beyond statins and PCSK9 (proprotein convertase subtilisin/kexin type 9) inhibitors.

Timing Matters: Late, but Not Early, Exercise Training Ameliorates MASLD in Part by Modulating the Gut-Liver Axis in Mice.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects two billion people worldwide and is currently mostly treatable via lifestyle interventions, such as exercise training. However, it is unclear whether the positive effects of exercise are restricted to unique circadian windows. We therefore aimed to study whether the timing of exercise training differentially modulates MASLD development. Twenty weeks old male APOE*3-Leiden.CETP mice were fed a high fat-high cholesterol diet to induce MASLD and treadmill-trained for 1 h five times per week for 12 weeks either early (ZT13; E-RUN) or late (ZT22; L-RUN) in the dark phase while corresponding sedentary groups (E-SED and L-SED) did not. Late, but not early exercise training decreased the MASLD score, body weight, fat mass, and liver triglycerides, accompanied by an altered composition of the gut microbiota. Specifically, only late exercise training increased the abundance of short-chain fatty acid-producing bacterial families and genera, such as Akkermansia, Lachnospiraceae, and Rikenella. To assess the role of the gut microbiota in training-induced effects, the study was repeated and trained (ZT22 only, RUN) or sedentary mice (SED) served as fecal donors for sedentary recipient mice (RUN FMT and SED FMT). Fecal microbiota transplantation reduced liver weight and plasma triglycerides in RUN FMT compared to SED FMT and tended to lower the MASLD score and liver triglycerides. Timing of exercise training is a critical factor for the positive effect on MASLD in this preclinical model, and the effect of late exercise is partially mediated via the gut-liver axis.

Toxicity and efficacy study of a combination of two retinoic acids in an ApoE knockout mouse model of atherosclerosis.

Atherosclerosis is a major contributor to cardiovascular disease, characterized by inflammation and lipid accumulation in arterial walls, leading to plaque formation. Elevated low-density lipoprotein cholesterol is a primary risk factor for atherosclerosis. All-trans retinoic acid (ATRA), a metabolite of vitamin A, has demonstrated anti-inflammatory effects and potential in regulating vascular injury. 9-cisretinoic acid (9cRA) is an active metabolite of vitamin A and activates the retinoid X receptor. This study investigates whether potassium retinoate (PA9RA), a synthetic combination of ATRA and 9cRA, offers superior efficacy in treating atherosclerosis compared to established treatments such as clopidogrel and atorvastatin. Male ApoE-/- mice were fed a Western-type diet and treated with PA9RA, clopidogrel, or atorvastatin for 10 weeks. The body weight, organ weight, serum biochemistry, and histopathology, including atherosclerotic lesion area and liver steatosis were assessed. PA9RA treatment led to a significant reduction in body weight and inguinal fat, with the 45 mg/kg/day dose showing marked efficacy in decreasing atherosclerotic lesion size and ameliorating liver steatosis. Histopathological evaluation revealed decreased foam cell formation and improved liver histology in PA9RA-treated groups compared to controls. Notable side effects included epidermal hyperplasia and gastric hyperplasia at high doses of PA9RA. PA9RA exhibits superior efficacy over clopidogrel and atorvastatin in ameliorating atherosclerosis and fatty liver in ApoE-/- mice. This study highlights PA9RA's potential as a promising therapeutic agent for atherosclerosis. Further research is needed to elucidate its mechanisms of action and assess long-term safety and efficacy.

Inhibition of ceramide de novo synthesis mitigates atherosclerosis

Abstract Background Atherosclerosis is the primary underlying cause of cardiovascular disease. Plasma ceramides are positively correlated with an increased risk of major adverse cardiovascular events and serve as an alternative biomarker for cardiovascular disease. Importantly, ceramide levels are notably higher within atherosclerotic plaques. Therefore, inhibiting the ceramide de novo synthesis pathway in the plaques may serve as a potential therapeutic intervention for atherosclerosis. Purpose We aim to assess the therapeutic efficacy of a nano-formulation of myriocin, a targeted inhibitor of serine-palmitoyl transferase, the key enzyme in ceramide de novo synthesis, in treating atherosclerosis. Methods Myriocin was encapsulated within the hydrophobic core of lipid-based nanoparticles specifically designed for this study. Male and female ApoE-/- mice, subjected to an 8-week high-fat diet regimen, received either myriocin nanoparticles (treatment group) or empty nanoparticles (control group) intravenously once a week for 4 weeks at a dose of 1.3 mg/kg body weight while still on the high-fat diet. At the study endpoint, whole aortas were harvested and subjected to Oil Red O staining to quantify the total lesion area. Histological staining was performed on serial sections of the aortic root to analyze the cellular composition within the plaques. Plasma samples were collected for total cholesterol measurement and lipidomic analysis. Results Upon intravenous administration, myriocin nanoparticles exhibited preferential accumulation in atherosclerotic plaques. Compared to control mice, those treated with myriocin nanoparticles exhibited a 58.7% reduction in total lesion area in male ApoE-/- mice and a 64.6% reduction in female ApoE-/- mice (p<0.0001). Consistently, the lesion areas in the aortic root were also reduced in the treated mice. Notably, myriocin nanoparticles enhanced collagen and alpha-smooth muscle content, while reducing macrophages and neutral lipids within the plaques, leading to a reduced plaque vulnerability index and preventing rupture events. TUNEL staining revealed reduced cellular apoptosis in the atherosclerotic plaques, resulting in a smaller necrotic area in the treated mice. In vitro experiments demonstrated that myriocin nanoparticles effectively inhibited lipopolysaccharide-induced inflammatory response and oxidative stress in bone marrow-derived macrophages. Furthermore, myriocin nanoparticles restored the gene expression of enzymes involved in the fatty acid beta-oxidation pathway, such as ACOX1 and CPT1A, in foam cells. Conclusion Using myriocin nanoparticles to inhibit excessive ceramide production due to inflammation reduces inflammation, and ROS production, while restoring fatty acid beta-oxidation in macrophages. This approach emerges as an effective strategy for targeting ceramide de novo synthesis within atherosclerotic plaques, thereby mitigating atherosclerosis progression and improving plaque stability.

Combined administration of atorvastatin and mesenchymal stem cells synergizes to attenuate inflammation and promote efferocytosis in atherosclerotic mice

Abstract Background Atherosclerosis (AS), characterized by chronic systemic and lesional inflammation, is by far the most frequent underlying cause of cardio-cerebrovascular diseases. Recently, impaired efferocytosis, referring to defective removal of dead cells, was identified as another essential factor contributing to the development of AS, which is partly attributed to upregulation of CD47 and explain the benefits of atorvastatin (ATV). Mesenchymal stem cells (MSCs) were reported to protect against AS by inhibiting inflammation; while whether ATV and MSCs could synergize to attenuate inflammation and promote efferocytosis and consequently, reduce AS burden, remains unclear. Methods To establish the AS model, 6-week-old male ApoE-/- mice fed with Western diet for 12 weeks were used and grouped randomly (n=15 per group). The scheme of administration of ATV and human umbilical cord MSCs was shown in Figure 1A. After 6-week treatment, mice were sacrificed, and hearts and aortas were harvested. Oil Red O staining was used to evaluate the lesional burden. Immunofluorescence staining was used to assess the inflammation and efferocytosis in the plaque. A high level of CD11b+ cells (macrophages) infiltration was considered an indicator of inflammation; a high level of cleaved caspase 3 puncta unmerged with macrophages (Mac3+ area) was considered an indicator of impaired efferocytosis. Results Human umbilical cord MSCs were identified as CD73+CD90+CD105+CD45-CD14- cells. Evoked inflammation and defective clearance of apoptotic cells in the plaque were observed (Figure 1B, C). ATV and MSC treatment inhibited macrophage infiltration and activated efferocytosis. Given the mechanistic idea that upregulated CD47 blocks the Signal regulatory protein α (SIRPα) / src homology-2 (SH2)-domain-containing protein tyrosine phosphatases (SHP) signal and leads to efferocytosis impairment, we evaluated the expression levels of CD47 in plaque and phosphorylated SHP (p-SHP) in lesional macrophages (Figure 2A, B). The elevation of CD47 expression and consequent downregulation of p-SHP was reversed by both ATV and MSCs, with a lower lesion burden observed in NS+MSC, ATV+PBS and ATV+MSC groups. All benefits were more pronounced in the group received combined treatment (ATV+MSC). Conclusions The results indicated ATV and MSC administration could synergizes to attenuate inflammation and promote efferocytosis in AS plaque, and the combined treatment led to the lowest lesion burden. CD47/p-SHP signal plays an essential role in the activation of efferocytosis. The mechanisms underlying the inhibition of CD47 are currently under investigation.

Toll-Like Receptor 2 Attenuates the Formation and Progression of Angiotensin II-Induced Abdominal Aortic Aneurysm in ApoE−/− Mice

Introduction: We demonstrated Toll-like receptor (TLR) 4 in the pathogenesis of angiotensin II (AngII)-mediated abdominal aortic aneurysm (AAA) formation. Here, we study TLR2 in the AAA formation. Methods: Male ApoE−/− and ApoE−/−TLR2−/− mice were treated with AngII. Mice were injected with the TLR2 agonist Pam3CSK4. The incidence and severity of AAA were determined. MCP-1, MCP-5, RANTES, CXCL10, CCR5, and CXCR3 were analyzed. M1 and M2 macrophages in the aorta were detected by flow cytometry. Results: These studies demonstrated an increase in AAA formation in TLR2−/− mice and a decrease by Pam3CSK4. Pam3CSK4 decreased the ratio of M1/M2 and the levels of RANTES, CXCL10, CCR5, and CXCR3. Furthermore, Pam3CSK4 treatment 1 week following AngII retarded the progression of AAA. Conclusion: These data demonstrated a protective effect of TLR2 signaling on AAA in association with a decrease in the ratio of M1 to M2 macrophages and the expression of chemokines and their receptors. Furthermore, the treatment of Pam3CSK4 after AngII demonstrated a marked retardation of lesion progression. Given the fact that most AAA patients are detected late in the disease process, these findings suggest that TLR2 stimulation may play a therapeutic role in retarding disease progression.

Nuciferine Alleviates High-Fat Diet- and ApoE-/--Induced Hepatic Steatosis and Ferroptosis in NAFLD Mice via the PPARα Signaling Pathway.

Nonalcoholic fatty liver disease (NAFLD) causes significant global mortality and healthcare costs with no recommended pharmacological intervention for clinical management. Nuciferine (Nuc) is an alkaloid with aromatic rings, abundantly found in Nelumbo nucifera Gaertn. In this study, we explored the protective mechanisms of Nuc against hepatic steatosis and ferroptosis in NAFLD. High-fat diet (HFD) and healthy male ApoE-/- mice were used to induce NAFLD and a hypercholesterolemia model. Nuc was administered to the mice for four consecutive weeks from the ninth week. Various assessments, including histopathology, RNA sequencing, lipid metabolism, and ferroptosis-related protein expression, showed that Nuc alleviated hepatic steatosis and ferroptosis. We further showed that Nuc improves fatty acid accumulation and ferroptosis through the PPARα signaling pathway in mice and RSL3-treated AML-12 cells. The PPARα inhibitor GW6471 blocked Nuc's protective effects, leading to excess accumulation of iron ions. Thus, Nuc may be a potential therapeutic agent for NAFLD.

CCN4 (WISP-1) reduces apoptosis and atherosclerotic plaque burden in an ApoE mouse model

CCN4/WISP-1 regulates various cell behaviours that contribute to atherosclerosis progression, including cell adhesion, migration, proliferation and survival. We therefore hypothesised that CCN4 regulates the development and progression of atherosclerotic plaques. We used a high fat fed ApoE-/- mouse model to study atherosclerotic plaque progression in the brachiocephalic artery and aortic root. In protocol 1, male ApoE-/- mice with established plaques were given a CCN4 helper-dependent adenovirus to see the effect of treatment with CCN4, while in protocol 2 male CCN4-/-ApoE-/- were compared to CCN4+/+ApoE-/- mice to assess the effect of CCN4 deletion on plaque progression. CCN4 overexpression resulted in reduced occlusion of the brachiocephalic artery with less apoptosis, fewer macrophages, and attenuated lipid core size. The amount of plaque found on the aortic root was also reduced. CCN4 deficiency resulted in increased apoptosis and occlusion of the brachiocephalic artery as well as increased plaque in the aortic root. Additionally, in vitro cells from CCN4-/-ApoE-/- mice had higher apoptotic levels. CCN4 deficiency did not significantly affect blood cholesterol levels or circulating myeloid cell populations. We conclude that in an atherosclerosis model the most important action of CCN4 is the effect on cell apoptosis. CCN4 provides pro-survival signals and leads to reduced cell death, lower macrophage number, smaller lipid core size and reduced atherosclerotic plaque burden. As such, the pro-survival effect of CCN4 is worthy of further investigation, in a bid to find a therapeutic for atherosclerosis.

Mesenchymal stromal cell-derived exosomes protect against abdominal aortic aneurysm formation through CD74 modulation of macrophage polarization in mice

BackgroundMesenchymal stromal cell (MSC)-derived exosomes (MSC-Exo) have been recognized for their significant role in regulating macrophage polarization, a process crucial to the pathogenesis of abdominal aortic aneurysm (AAA). However, the therapeutic effects of MSC-Exo on AAA remain largely unexplored. Therefore, this study aimed to investigate the functional and mechanistic aspects of MSC-Exo in the progression of AAA.MethodsThe MSC-derived exosomes were characterized using Transmission Electron Microscopy, Nanoparticle Tracking Analysis, and Western blotting. An experimental mouse model of AAA was established through the administration of angiotensin II (Ang II) in male apoe−/− mice and calcium chloride (CaCl2) in male C57/B6 mice, with subsequent tail vein injection of exosomes to evaluate their efficacy against AAA. Macrophage polarization was assessed using immunofluorescence staining and WB analysis. Mechanistic analysis was performed using 4D Label-free Proteomics analysis.ResultsWe found that intravenous administration of MSC-Exo induced M2 polarization of macrophages within an inflammatory environment, effectively impeding AAA development in Ang II or CaCl2-induced AAA model. The therapeutic efficacy of MSC-Exo treatment was dependent on the presence of macrophages. Mechanistically, MSC-Exo suppressed the levels of cluster of differentiation 74 (CD74), modulating macrophage polarization through the TSC2-mTOR-AKT pathway. These findings highlight the potential of MSC-Exo as a therapeutic strategy for AAA by modulating macrophage polarization.

Enhanced atherosclerosis in apolipoprotein E knockout rabbits: role of apoB48-rich remnant lipoproteins.

Apolipoprotein E (apoE) acts as a binding molecule for both the low-density lipoprotein receptor and the lipoprotein receptor-related protein and this function is essential for facilitating the hepatocyte uptake of lipoproteins containing apoB. The absence of apoE leads to increased atherogenicity in both humans and mice, although the precise molecular mechanisms remain incompletely understood. This study aimed to investigate the susceptibility of apoE knockout (KO) rabbits, in comparison with wild-type (WT) rabbits, to diet-induced hyperlipidemia and atherosclerosis. ApoE KO rabbits and WT rabbits were fed a diet containing 0.3% cholesterol for 16 weeks. Plasma lipid levels, lipoproteins, and apolipoproteins were analyzed. Atherosclerosis was evaluated at the endpoint of experiments. In addition, we evaluated the oxidizability of those lipoproteins containing apoB to investigate the possible mechanisms of atherosclerosis. Male apoE KO rabbits showed significantly elevated levels of total cholesterol and triglycerides compared to WT rabbits, while female apoE KO rabbits displayed similar high total cholesterol levels, albeit with significantly higher triglycerides levels than WT controls. Notably, both male (2.1-fold increase) and female (1.6-fold increase) apoE KO rabbits exhibited a significantly augmented aortic lesion area compared to WT controls. Pathological examination showed that the increased intimal lesions in apoE KO rabbits were featured by heightened infiltration of macrophages (2.7-fold increase) and smooth muscle cells (2.5-fold increase). Furthermore, coronary atherosclerotic lesions were also increased by 1.3-fold in apoE KO rabbits. Lipoprotein analysis revealed that apoB48-rich beta-very-low-density lipoproteins were notably abundant in apoE KO rabbits, suggesting that these remnant lipoproteins of intestinal origin serve as the primary atherogenic lipoproteins. Moreover, apoB48-rich remnant lipoproteins isolated from apoE KO rabbits exhibited heightened susceptibility to copper-induced oxidation. The findings indicate that apoB48-rich remnant lipoproteins, resulting from apoE deficiency, possess greater atherogenic potential than apoB100-rich remnant lipoproteins, regardless of plasma TC levels.

The Anti-Atherosclerotic Effects of Endothelin Receptor Antagonist, Bosentan, in Combination with Atorvastatin-An Experimental Study.

Bosentan, an endothelin receptor antagonist (ERA), has potential anti-atherosclerotic properties. We investigated the complementary effects of bosentan and atorvastatin on the progression and composition of the atherosclerotic lesions in diabetic mice. Forty-eight male ApoE-/- mice were fed high-fat diet (HFD) for 14 weeks. At week 8, diabetes was induced with streptozotocin, and mice were randomized into four groups: (1) control/COG: no intervention; (2) ΒOG: bosentan 100 mg/kg/day per os; (3) ATG: atorvastatin 20 mg/kg/day per os; and (4) BO + ATG: combined administration of bosentan and atorvastatin. The intra-plaque contents of collagen, elastin, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-a (TNF-a), matrix metalloproteinases (MMP-2, -3, -9), and TIMP-1 were determined. The percentage of lumen stenosis was significantly lower across all treated groups: BOG: 19.5 ± 2.2%, ATG: 12.8 ± 4.8%, and BO + ATG: 9.1 ± 2.7% compared to controls (24.6 ± 4.8%, p < 0.001). The administration of both atorvastatin and bosentan resulted in significantly higher collagen content and thicker fibrous cap versus COG (p < 0.01). All intervention groups showed lower relative intra-plaque concentrations of MCP-1, MMP-3, and MMP-9 and a higher TIMP-1concentration compared to COG (p < 0.001). Importantly, latter parameters presented lower levels when bosentan was combined with atorvastatin compared to COG (p < 0.05). Bosentan treatment in diabetic, atherosclerotic ApoE-/- mice delayed the atherosclerosis progression and enhanced plaques' stability, showing modest but additive effects with atorvastatin, which are promising in atherosclerotic cardiovascular diseases.

High-fat-diet-induced obesity promotes simultaneous progression of lung cancer and atherosclerosis in apolipoprotein E-knockout mice.

Clinical studies have shown that atherosclerotic cardiovascular disease and cancer often co-exist in the same individual. The present study aimed to investigate the role of high-fat-diet (HFD)-induced obesity in the coexistence of the two diseases and the underlying mechanism in apolipoprotein E-knockout (ApoE-/-) mice. Male ApoE-/- mice were fed with a HFD or a normal diet (ND) for 15 weeks. On the first day of Week 13, the mice were inoculated subcutaneously in the right axilla with Lewis lung cancer cells. At Weeks 12 and 15, serumlectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and vascular endothelial growth factor levels were measured by enzyme-linked immunosorbent assay, and blood monocytes and macrophages were measured by fluorescence-activated cell sorting. At Week 15, the volume and weight of the local subcutaneous lung cancer and metastatic lung cancer and the amount of aortic atherosclerosis were measured. At Week 15, compared with mice in the ND group, those in the HFD group had a larger volume of local subcutaneous cancer (p = 0.0004), heavier tumors (p = 0.0235), more metastatic cancer in the lungs (p < 0.0001), a larger area of lung involved in metastatic cancer (p = 0.0031), and larger areas of atherosclerosis in the aorta (p < 0.0001). At Week 12, serum LOX-1, serum vascular endothelial growth factor, and proportions of blood monocytes and macrophages were significantly higher in the HFD group than those in the ND group (p = 0.0002, p = 0.0029, p = 0.0480, and p = 0.0106, respectively); this trend persisted until Week 15 (p = 0.0014, p = 0.0012, p = 0.0001, and p = 0.0204). In this study, HFD-induced obesity could simultaneously promote progression of lung cancer and atherosclerosis in the same mouse. HFD-induced upregulation of LOX-1 may play an important role in the simultaneous progression of these two conditions via the inflammatory response and VEGF.

The novel bispecific anti-NLRP3 inflammasome antibody InflamAb inhibits atherosclerosis in apolipoprotein E-deficient mice

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Dutch Heart Foundation Background/Introduction Atherosclerosis is the underlying pathology of many cardiovascular diseases and is characterized by chronic inflammation in the larger arteries. Activation of the NLRP3 inflammasome is one of the drivers of inflammation during atherosclerosis. Purpose Therefore, we aimed to determine the ability of the novel bispecific anti-NLRP3 inflammasome antibody, called InflamAb, to inhibit atherosclerosis. Methods and Results In vitro treatment of bone marrow derived macrophages with 25 ng/mL InflamAb effectively inhibited the IL-1β release induced by NLRP3 inflammasome activation with LPS and Aluminium hydroxide (P&amp;lt;0.05). In addition, InflamAb administration in western-type diet fed Apoe-/- mice significantly reduced circulating IL-1β at 4 hours post inflammasome activation (P&amp;lt;0.05), thereby showing in vivo efficacy of InflamAb. Subsequently, we assessed the effect of InflamAb on both the development of atherosclerosis and on pre-existing plaques. Treatment of western-type diet fed female Apoe-/- mice with 100 µg InflamAb or isotype control antibody (3x per week i.p) significantly inhibited collar-induced atherosclerotic plaque development in the carotid artery from 59±8*10^3 µm^2 in control mice to 36±5*10^3 µm^2 (P&amp;lt;0.05), which coincided with a reduction in relative macrophage (control: 36±2% versus InflamAb: 28±3%, P&amp;lt;0.05) and necrotic core content (control: 16±2% versus InflamAb: 8±1%, P&amp;lt;0.05). InflamAb treatment of male Apoe-/- with pre-existing atherosclerosis did not affect lesion size, but improved plaque stability parameters as illustrated by a reduced relative macrophage (control: 48±2% versus InflamAb: 42±2%, P&amp;lt;0.05) and necrotic core (control: 21±1% versus InflamAb: 18±1%, P&amp;lt;0.05) content. In addition, we observed a trend towards increased collagen levels upon InflamAb treatment (control: 39±2% versus InflamAb: 44±2%, P=0.08). Conclusion To conclude, NLRP3 inflammasome inhibition by the bispecific antibody InflamAb shows promising efficacy in inhibiting atherosclerotic plaque development and destabilization.

Treg Immunomodulation Contributes to the Anti-atherosclerotic Effects of Huxin Formula in ApoE-/- Mice.

To explore the effects of Huxin formula (HXF) in curtailing atherosclerosis and its underlying mechanism. According to random number table method, 24 specific pathogen free male ApoE-/- mice were randomly divided into model group, HXF low-dose (HXF-L) group (8.4 g/kg daily), HXF high-dose (HXF-H) group (16.8 g/kg daily), and pravastatin (8 mg/kg daily) group in Experiment I (n=6 per group). C57BL/6J mice served as the control group (n=6). ApoE-/- mice in HXF-L, HXF-H, pravastatin groups were fed a Western diet and administered continuously by gavage for 12 weeks, while C57BL/6J mice in the control group were fed conventional lab mouse chow for 12 weeks. Further, Tregs were depleted by weekly intraperitoneal injection of purified anti-mouse CD25 antibody (PC61, 250 µg per mouse) for 4 weeks in Experiment II (n=6 per group). Oil Red O and Masson staining were used to evaluate the plaque area and aortic root fibrosis. The CD4+CD25+Foxp3+Treg counts in the lymph nodes and spleen cells were detected using flow cytometric analysis. The transforming growth factor-β1 (TGF-β1), interleukin (IL)-10, and IL-6 serum levels were examined by MILLIPLEX® MAP technology. Quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot were utilized to assess the expression of TGF-β mRNA and protein in the aorta. The expression of CD4+T lymphocytes, macrophages and smooth muscle cells in the aortic root were detected by immunofluorescence staining. HXF reduced plaque area in ApoE-/- mice (P<0.01). HXF increased the Treg counts in the lymph nodes and spleen cells (P<0.05 or P<0.01). Moreover, HXF alleviated inflammatory response via elevating IL-10 and TGF-β 1 serum levels (P<0.05), while decreasing the IL-6 serum levels in ApoE-/- mice (P>0.05). Also, HXF upregulated the expression of TGF-β mRNA and protein in the aorta (P<0.05). Additionally, HXF attenuated CD4+T lymphocytes, macrophages and smooth muscle cells in aortic root plaque (P<0.01). Furthermore, the depletion of Tregs with CD25 antibody (PC61) curtailed the reduction in plaque area and aortic root fibrosis by HXF (P<0.01). HXF relieved atherosclerosis, probably by restraining inflammatory response, reducing inflammatory cell infiltration and attenuating aortic root fibrosis by increasing Treg counts.

Workflow for the unsupervised clustering of sleep stages identifies light and deep sleep in electrophysiological recordings in mice

Sleep physiology plays a critical role in brain development and aging. Accurate sleep staging, which categorizes different sleep states, is fundamental for sleep physiology studies. Traditional methods for sleep staging rely on manual, rule-based scoring techniques, which limit their accuracy and adaptability. We describe, test and challenge a workflow for unsupervised clustering of sleep states (WUCSS) in rodents, which uses accelerometer and electrophysiological data to classify different sleep states. WUCSS utilizes unsupervised clustering to identify sleep states using six features, extracted from 4-second epochs. We gathered high-quality EEG recordings combined with accelerometer data in diverse transgenic mouse lines (male ApoE3 versus ApoE4 knockin; male CNTNAP2 KO versus wildtype littermates). WUCSS showed high recall, precision, and F1-score against manual scoring on awake, NREM, and REM sleep states. Within NREM, WUCSS consistently identified two additional clusters that qualify as deep and light sleep states. The ability of WUCSS to discriminate between deep and light sleep enhanced the precision and comprehensiveness of the current mouse sleep physiology studies. This differentiation led to the discovery of an additional sleep phenotype, notably in CNTNAP2 KO mice, showcasing the method's superiority over traditional scoring methods. WUCSS, with its unsupervised approach and classification of deep and light sleep states, provides an unbiased opportunity for researchers to enhance their understanding of sleep physiology. Its high accuracy, adaptability, and ability to save time and resources make it a valuable tool for improving sleep staging in both clinical and preclinical research.

Abstract 1001: Interleukin-33 Deficiency Exacerbated Angiotensin II-induced Abdominal Aortic Aneurysms

Objective: Chronic infusion of angiotensin II (AngII) promotes development of abdominal aortic aneurysms (AAAs) in Apolipoprotein E-deficient (ApoE-/-) mice. Interleukin 33 (IL-33), known as one of IL-1 family, is rapidly released from damaged endothelial and epithelial cells. IL-33 binds to its receptor, ST2, and exerts various physiological functions through its downstream signaling. Previous studies have shown that exogenous IL-33 attenuates development of AAAs through wound healing resolution. The purpose of this study was to evaluate whether IL-33 deficiency exacerbated development of AAAs by using IL-33xApoE double deficient (IL-33-/-xApoE-/-) mice. Methods and Results: Male ApoE-/- and IL-33-/-xApoE-/- (8-12 weeks old) mice were assigned into saline infused group (ApoE-/- n=5, IL-33-/-xApoE-/- n=7) and AngII infused group (ApoE-/- n=19, IL-33-/-xApoE-/- n=19). Mice were infused subcutaneously with either saline or AngII by osmotic minipumps for 4 weeks. During 4-week AngII infusion, 3 mice died by AAA rupture in each AngII-infused group. There was no significant difference in ex vivo abdominal aortic width between saline infused groups (ApoE-/- 0.87±0.17 mm vs IL-33-/-xApoE-/- 0.87±0.14 mm, n.s.) nor body weight (ApoE-/- 27.3±0.9 g vs IL-33-/-xApoE-/- 27.9±0.9 g, n.s.) but serum total cholesterol concentrations in IL-33-/-xApoE-/- was higher than in ApoE-/- (522±85 mg/dL vs 427±108 mg/dL, p=0.026). AngII equally elevated systolic blood pressure (ApoE-/- 151±15 mmHg, IL-33-/-xApoE-/- 155±17 mmHg, n.s.). AngII increased ex vivo maximum abdominal aortic width in AngII infused groups, but AAAs were further exacerbated in the group of IL-33-/-xApoE-/- mice (ApoE-/- 1.31±0.09 mm vs IL-33-/-xApoE-/- 1.76±0.09 mm, p=0.001). In addition, the severity of AAAs in AngII infused groups was significantly worse in IL-33-/-xApoE-/- than in ApoE-/- (p=0.037). Conclusion: Our study demonstrated IL-33 deficiency exacerbated AngII-induced AAAs in male ApoE-/- mice.

Characterization of Endothelial to Mesenchymal Transition in Air Pollution Mediated Atherosclerosis: Insights from Trajectory Inference and Functional Analyses in Single cell RNA Sequencing Data

Background: Air pollution is the world’s leading environmental risk factor for atherosclerotic cardiovascular disease (ASCVD). Regressive transcriptional reprogramming and transition of cells to primordial states has been identified as a driver of atherosclerosis. Endothelial cell to mesenchymal cell transition (EndoMT) whereby an endothelial cell transforms to a mesenchymal-like cellular phenotype is one such mechanism. However its relevance to air pollution exposure remains uncharacterized. Methods: Male ApoE−/− mice were inhalationally exposed to concentrated particulate matter &lt;2.5μ component (PM2.5) or filtered air (FA, controls) using a Versatile Aerosol Concentration Enrichment System (VACES, 5–6 h/day, 5 d/week for 36 weeks, n = 7/group. PM2.5 concentrations were 70-80 μg/m3 or ~10X ambient levels. Following euthanasia, single-cell RNA-sequencing of aortic cells was performed using the 10-X platform (CA). For all subsequent analyses, aortic cell samples were integrated. Unsupervised clustering (k-NN) and low-dimensional space visualization (UMAP) were performed with a supervised graph method (Minimum Spanning Tree and Principal Curves) to infer and visualize trajectory analysis of novel cell lineages (branching and cell ordering in pseudotime/pseudospace). Functional analyses were carried out by statistical enrichment analysis using Over Representation Analysis (ORA) and Gene Set Enrichment Analysis (GSEA) against databases of Gene Ontologies (GO), Biological Pathways, putative regulatory motifs, proteins, or disease annotations to find over-representation of terms and functions. Pseudotime distribution assessed along exposure groups and cell-types lineages were statistically compared using GLM models. Differential Expression Analysis within lineages and between groups were tested by GLM and GAM models and FDR correction, followed by functional analyses. Results: Comparison of the pseudotime lineage with lineage root node at the endothelial cells revealed a similarity between observed endothelial (EC) to vascular smooth muscle (VSMC) cell transition with the differential transition noted with PM2.5 from FA. This overlap suggests a potential exaggeration of EC to VSMC transition in response to PM2.5 exposure. Differential gene expression profiling upon PM2.5-exposure in EC revealed significant changes in GO terms related to cell differentiation, cell migration, and regulation of cell population proliferation. Specific genes with variable patterns of expression (4481) were identified along the EC to VSMC lineage. KEGG pathway analysis along the lineage revealed enriched pathways including PI3K/Akt signaling pathway, MAPK signaling pathway, and Rap1 signaling pathway which are all implicated in the development and progression of atherosclerosis. Conclusion: Single-cell analysis reveals that chronic exposure to PM2.5 results in the exaggeration of EC to VSMC transition consistent with the EndoMT paradigm that may help explain the acceleration of ASCVD in response to air pollution exposure. Additional analysis of cell specific pathways may provide improved understanding of mechanisms progression of atherosclerosis in response to environmental exposures. NIH: 1R35ES031702-01, R01 HL155450, 3R35ES031702-02S1; Burroughs Wellcome Fund: 1060485. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.