Low-density Lipoprotein Receptor Knockout Mice Research Articles

Dihydromyricetin Alleviates Nonalcoholic Fatty Liver Disease and Its Associated Metabolic Syndrome by Inhibiting Endoplasmic Reticulum Stress in LDLR−/− Mice Fed with a High-Fat and High-Fructose Diet

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease. Previous studies have shown that dihydromyricetin (DHM) is beneficial for NAFLD. However, whether DHM alleviates NAFLD by inhibiting liver endoplasmic reticulum (ER) stress remains unknown. Thus, this study aimed to identify the potential roles and mechanisms of DHM. Twenty-four male low-density lipoprotein receptor (LDLR−/−) knockout mice aged 8 weeks were randomly divided into normal control, control, and DHM groups. Normal control mice were fed a normal diet (ND), and the last two groups of mice were fed a high-fat and high-fructose diet (HFD) for 12 weeks, treated with or without DHM. DHM alleviated diet-induced hyperlipidemia as early as 4 weeks after and until the end of HFD feeding. HFD increased insulin resistance, and the opposite was observed in the DHM group. Compared to the control group, the body weight of the mice and adipocyte size and weight of the retroperitoneal and epididymal fat were remarkably reduced in the DHM group. The expression of genes related to lipid metabolism, such as Acox1 and Cpt1α, was significantly upregulated. Moreover, Mttp was downregulated in the two fat sits in the DHM group. DHM alleviated diet-induced lipid deposition in the liver and decreased liver triglyceride and total cholesterol content. DHM improved liver function by inhibiting ER stress, alleviating atherogenesis, and promoting vascular remodeling. In conclusion, dihydromyricetin improved NAFLD and related insulin resistance, hyperlipidemia, and atherogenesis by inhibiting liver ER stress in HFD-fed LDLR−/− mice.

The Beneficial Effect of Lomitapide on the Cardiovascular System in LDLr-/- Mice with Obesity.

Homozygous familial hypercholesteremia (HoFH) is a rare, life-threatening metabolic disease, mainly caused by a mutation in the LDL receptor. If untreated, HoFH causes premature death from acute coronary syndrome. Lomitapide is approved by the FDA as a therapy to lower lipid levels in adult patients with HoFH. Nevertheless, the beneficial effect of lomitapide in HoFH models remains to be defined. In this study, we investigated the effect of lomitapide on cardiovascular function using LDL receptor-knockout mice (LDLr-/-). Six-week-old LDLr-/- mice were fed a standard diet (SD) or a high-fat diet (HFD) for 12 weeks. Lomitapide (1 mg/Kg/Day) was given by oral gavage for the last 2 weeks in the HFD group. Body weight and composition, lipid profile, blood glucose, and atherosclerotic plaques were measured. Vascular reactivity and markers for endothelial function were determined in conductance arteries (thoracic aorta) and resistance arteries (mesenteric resistance arteries (MRA)). Cytokine levels were measured by using the Mesoscale discovery V-Plex assays. Body weight (47.5 ± 1.5 vs. 40.3 ± 1.8 g), % of fat mass (41.6 ± 1.9% vs. 31.8 ± 1.7%), blood glucose (215.5 ± 21.9 vs. 142.3 ± 7.7 mg/dL), and lipid levels (cholesterol: 600.9 ± 23.6 vs. 451.7 ± 33.4 mg/dL; LDL/VLDL: 250.6 ± 28.9 vs. 161.1 ± 12.24 mg/dL; TG: 299.5 ± 24.1 vs. 194.1 ± 28.1 mg/dL) were significantly decreased, and the % of lean mass (56.5 ± 1.8% vs. 65.2 ± 2.1%) was significantly increased in the HFD group after lomitapide treatment. The atherosclerotic plaque area also decreased in the thoracic aorta (7.9 ± 0.5% vs. 5.7 ± 0.1%). After treatment with lomitapide, the endothelium function of the thoracic aorta (47.7 ± 6.3% vs. 80.7 ± 3.1%) and mesenteric resistance artery (66.4 ± 4.3% vs. 79.5 ± 4.6%) was improved in the group of LDLr-/- mice on HFD. This was correlated with diminished vascular endoplasmic (ER) reticulum stress, oxidative stress, and inflammation. Treatment with lomitapide improves cardiovascular function and lipid profile and reduces body weight and inflammatory markers in LDLr-/- mice on HFD.

Glial cell reactivity drives cognitive decline in LDLr‐/‐ mice: experimental evidences

AbstractBackgroundFamilial hypercholesterolemia (FH) is a clinical condition characterized by increased plasma cholesterol levels, due to a mutation in the gene encoding the low‐density lipoprotein receptor (LDLr) in cells [Brown and Goldstein, 1984]. Importantly, clinical evidence indicates that FH negatively affects cognition and favors the development of neuropathologies [Ariza et al., 2016; Zambón et al, 2010], however, the mechanisms behind this relationship are still not fully understood. In this study we aimed to investigate the role of glial cell reactivity on the cognitive deficits related to the FH, as well as the possible neuroprotective effect of a pharmacological inhibitor of microglial cell activation.MethodFirstly, male 3‐months‐old and 14‐months‐old C57BL/6 wild‐type and LDLr knockout mice (LDLr‐/‐) mice brains were prepared for the evaluation of glial cell morphology by immunofluorescence. In addition, the mice’s hippocampus was removed to measure the immune content of synaptic and tight‐junctions proteins. Moreover, male and female 6‐months‐old C57BL/6 wild‐type and LDLr‐/‐ mice were treated with minocycline (50 mg/kg) or vehicle (saline) for four weeks. Afterwards, mice were submitted to behavioral tests.ResultMiddle‐aged LDLr‐/‐ mice (14‐months‐old) exhibited increased microgliosis in hippocampus, which was correlated with morphological change of microglial cells and astrocytes in this same brain region. Also, middle‐aged LDLr‐/‐ mice presented a reduction in the immune content of synaptophysin‐1 in the hippocampus. Moreover, young adult (3‐months‐old) LDLr‐/‐ mice exhibited reduced immune content of PSD95 in the hippocampus. Finally, the administration of minocycline to 6‐months‐old LDLr‐/‐ mice ameliorated the performance of hypercholesterolemic mice in hippocampal‐dependent memory tasks, without modifying their locomotor activity and metabolic parameters.ConclusionTaken together, our results demonstrated that FH alters the glial cell reactivity, which was associated with changes in synaptic proteins in the hippocampus. In addition, the pharmacological modulation of microglial cell reactivity ameliorated the hippocampal‐dependent memory in LDLr‐/‐ mice, suggesting that increased glial cell reactivity is a key event for HF‐related cognitive deficits.

Carbon Nanodots Attenuate Lipid Peroxidation in the LDL Receptor Knockout Mouse Brain.

Abnormal cholesterol metabolism can lead to oxidative stress in the brain. Low-density lipoprotein receptor (LDLr) knockout mice are models for studying altered cholesterol metabolism and oxidative stress onset in the brain. Carbon nanodots are a new class of carbon nanomaterials that possess antioxidant properties. The goal of our study was to evaluate the effectiveness of carbon nanodots in preventing brain lipid peroxidation. LDLr knockout mice and wild-type C57BL/6J mice were treated with saline or 2.5 mg/kg bw of carbon nanodots for a 16-week period. Brains were removed and dissected into the cortex, midbrain, and striatum. We measured lipid peroxidation in the mouse brain tissues using the Thiobarbituric Acid Reactive Substances Assay and iron and copper concentrations using Graphite Furnace Atomic Absorption Spectroscopy. We focused on iron and copper due to their association with oxidative stress. Iron concentrations were significantly elevated in the midbrain and striatum of the LDLr knockout mice compared to the C57BL/6J mice, whereas lipid peroxidation was greatest in the midbrain and cortex of the LDLr knockout mice. Treatment with carbon nanodots in the LDLr knockout mice attenuated both the rise in iron and lipid peroxidation, but they had no negative effect in the C57BL/6J mice, indicating the anti-oxidative stress properties of carbon nanodots. We also assessed locomotor and anxiety-like behaviors as functional indicators of lipid peroxidation and found that treatment with carbon nanodots prevented the anxiety-like behaviors displayed by the LDLr knockout mice. Overall, our results show that carbon nanodots are safe and may be an effective nanomaterial for combating the harmful effects caused by lipid peroxidation.

Genetic or therapeutic neutralization of ALK1 reduces LDL transcytosis and atherosclerosis in mice

Low-density lipoprotein (LDL) accumulation in the arterial wall contributes to atherosclerosis initiation and progression1. Activin A receptor-like type 1 (ACVRL1, called activin-like kinase receptor (ALK1)) is a recently identified receptor that mediates LDL entry and transcytosis in endothelial cells (ECs)2,3. However, the role of this pathway in vivo is not yet known. In the present study, we show that genetic deletion of ALK1 in arterial ECs of mice substantially limits LDL accumulation, macrophage infiltration and atherosclerosis without affecting cholesterol or triglyceride levels. Moreover, a selective monoclonal antibody binding ALK1 efficiently blocked LDL transcytosis, but not bone morphogenetic protein-9 (BMP9) signaling, dramatically reducing plaque formation in LDL receptor knockout mice fed a high-fat diet. Thus, our results demonstrate that blocking LDL transcytosis into the endothelium may be a promising therapeutic strategy that targets the initiating event of atherosclerotic cardiovascular disease.

Tributyrin Intake Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm in LDLR-/- Mice.

Abdominal aortic aneurysm (AAA) is a multifactorial cardiovascular disease with a high risk of death, and it occurs in the infrarenal aorta with vascular dilatation. High blood pressure acts on the aortic wall, resulting in rupture and causing life-threatening intra-abdominal hemorrhage. Vascular smooth muscle cell (VSMC) dysregulation and extracellular matrix (ECM) degradation, especially elastin breaks, contribute to structural changes in the aortic wall. The pathogenesis of AAA includes the occurrence of oxidative stress, inflammatory cell infiltration, elastic fiber fragmentation, VSMC apoptosis, and phenotypic transformation. Tributyrin (TB) is decomposed by intestinal lipase and has a function similar to that of butyrate. Whether TB has a protective effect against AAA remains uncertain. In the present study, we established an AAA murine model by angiotensin II (AngII) induction in low-density lipoprotein receptor knockout (LDLR-/-) mice and investigated the effects of orally administered TB on the AAA size, ratio of macrophage infiltration, levels of matrix metalloproteinase (MMP) expression, and epigenetic regulation. TB attenuates AngII-induced AAA size and decreases elastin fragmentation, macrophage infiltration, and MMP expression in the medial layer of the aorta and reduces the levels of SBP (systolic blood pressure, p < 0.001) and MMP-2 (p < 0.02) in the serum. TB reduces the AngII-stimulated expression levels of MMP2 (p < 0.05), MMP9 (p < 0.05), MMP12, and MMP14 in human aortic smooth muscle cells (HASMCs). Moreover, TB and valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, suppress AngII receptor type 1 (AT1R, p < 0.05) activation and increase the expression of acetyl histone H3 by HDAC activity inhibition (p < 0.05). Our findings suggest that TB exerts its protective effect by suppressing the activation of HDAC to attenuate the AngII-induced AT1R signaling cascade.

DNA methylation changes underlie the long-term association between periodontitis and atherosclerotic cardiovascular disease.

Periodontitis, the leading cause of adult tooth loss, has been identified as an independent risk factor for cardiovascular disease (CVD). Studies suggest that periodontitis, like other CVD risk factors, shows the persistence of increased CVD risk even after mitigation. We hypothesized that periodontitis induces epigenetic changes in hematopoietic stem cells in the bone marrow (BM), and such changes persist after the clinical elimination of the disease and underlie the increased CVD risk. We used a BM transplant approach to simulate the clinical elimination of periodontitis and the persistence of the hypothesized epigenetic reprogramming. Using the low-density lipoprotein receptor knockout (LDLRo ) atherosclerosis mouse model, BM donor mice were fed a high-fat diet to induce atherosclerosis and orally inoculated with Porphyromonas gingivalis (Pg), a keystone periodontal pathogen; the second group was sham-inoculated. Naïve LDLR o mice were irradiated and transplanted with BM from one of the two donor groups. Recipients of BM from Pg-inoculated donors developed significantly more atherosclerosis, accompanied by cytokine/chemokines that suggested BM progenitor cell mobilization and were associated with atherosclerosis and/or PD. Using whole-genome bisulfite sequencing, 375 differentially methylated regions (DMRs) and global hypomethylation in recipients of BM from Pg-inoculated donors were observed. Some DMRs pointed to the involvement of enzymes with major roles in DNA methylation and demethylation. In validation assays, we found a significant increase in the activity of ten-eleven translocase-2 and a decrease in the activity of DNA methyltransferases. Plasma S-adenosylhomocysteine levels were significantly higher, and the S-adenosylmethionine to S-adenosylhomocysteine ratio was decreased, both of which have been associated with CVD. These changes may be related to increased oxidative stress as a result of Pg infection. These data suggest a novel and paradigm-shifting mechanism in the long-term association between periodontitis and atherosclerotic CVD.

Trib1 Deficiency Promotes Hyperlipidemia, Inflammation, and Atherosclerosis in LDL Receptor Knockout Mice.

Genetic variants at the TRIB1 gene locus are strongly associated with plasma lipid traits and the risk of coronary artery disease in humans. Here, we analyzed the consequences of Trib1 deficiency on lipid metabolism and atherosclerotic lesion formation in atherosclerosis-susceptible Ldlr-/- mice. Trib1-/- mice were crossed onto the Ldlr-/- background to generate double-knockout mice (Trib1-/-Ldlr-/-) and fed a semisynthetic, modified AIN76 diet (0.02% cholesterol and 4.3% fat) until 20 weeks of age. Trib1-/-Ldlr-/- mice had profoundly larger (5.8-fold) and more advanced atherosclerotic lesions at the aortic root as compared with Trib1+/+Ldlr-/- controls. Further, we observed significantly elevated plasma total cholesterol and triglyceride levels in Trib1-/-Ldlr-/- mice, resulting from higher VLDL (very-low-density lipoprotein) secretion. Lipidomics analysis revealed that loss of Trib1 altered hepatic lipid composition, including the accumulation of cholesterol and proinflammatory ceramide species, which was accompanied by signs of hepatic inflammation and injury. Concomitantly, we detected higher plasma levels of IL (interleukin)-6 and LCN2 (lipocalin 2), suggesting increased systemic inflammation in Trib1-/-Ldlr-/- mice. Hepatic transcriptome analysis demonstrated significant upregulation of key genes controlling lipid metabolism and inflammation in Trib1-/-Ldlr-/- mice. Further experiments suggested that these effects may be mediated through pathways involving a C/EPB (CCAAT/enhancer binding protein)-PPARγ (peroxisome proliferator-activated receptor γ) axis and JNK (c-Jun N-terminal kinase) signaling. We provide experimental evidence that Trib1 deficiency promotes atherosclerotic lesion formation in a complex manner that includes the modulation of lipid metabolism and inflammation.

PRMT5 inhibition induces pro-inflammatory macrophage polarization and increased hepatic triglyceride levels without affecting atherosclerosis in mice.

Protein arginine methyltransferase 5 (PRMT5) controls inflammation and metabolism through modulation of histone methylation and gene transcription. Given the important role of inflammation and metabolism in atherosclerotic cardiovascular disease, here we examined the role of PRMT5 in atherosclerosis using the specific PRMT5 inhibitor GSK3326595. Cultured thioglycollate-elicited peritoneal macrophages were exposed to GSK3326595 or DMSO control and stimulated with either 1ng/mL LPS or 100 ng/mL interferon-gamma for 24 h. Furthermore, male low-density lipoprotein (LDL) receptor knockout mice were fed an atherogenic Western-type diet and injected intraperitoneally 3×/week with a low dose of 5mg/kg GSK3326595 or solvent control for 9 weeks. In vitro, GSK3326595 primed peritoneal macrophages to interferon-gamma-induced M1 polarization, as evidenced by an increased M1/M2 gene marker ratio. In contrast, no difference was found in the protein expression of iNOS (M1 marker) and ARG1 (M2 marker) in peritoneal macrophages of GSK3326595-treated mice. Also no change in the T cell activation state or the susceptibility to atherosclerosis was detected. However, chronic GSK3326595 treatment did activate genes involved in hepatic fatty acid acquisition, i.e. SREBF1, FASN, and CD36 (+59%, +124%, and +67%, respectively; p< 0.05) and significantly increased hepatic triglyceride levels (+50%; p< 0.05). PRMT5 inhibition by low-dose GSK3326595 treatment does not affect the inflammatory state or atherosclerosis susceptibility of Western-type diet-fed LDL receptor knockout mice, while it induces hepatic triglyceride accumulation. Severe side effects in liver, i.e. development of non-alcoholic fatty liver disease, should thus be taken into account upon chronic treatment with this PRMT5 inhibitor.

8(R)-hydroxy-eicosapentaenoic acid (8R-HEPE) induces transcription of cholesterol efflux receptors via activation of liver X receptor in macrophages.

Dyslipidemia is a risk factor for the development of atherosclerotic cardiovascular disease. 8-HEPE from North Pacific krill (Euphausia pacifica) is known to reduce plasma LDL cholesterol levels and increase plasma HDL cholesterol levels in LDL receptor knock-out mice fed a western diet. Moreover, 8-HEPE also reduces the area of aortic atherosclerosis in apoE knock-out mice fed the same diet. In this study, we examined the stereochemical specific activity of 8-HEPE for inducing expression of cholesterol efflux receptors (Abca1 and Abcg1) in J774.1 cells. Our findings show 8R-HEPE induces the expression of Abca1 and Abcg1 via activation of liver X receptor, whereas 8S-HEPE elicits no such activity. These results suggest that 8R-HEPE derived from North Pacific krill may have beneficial effects against dyslipidemia.

Herpud1 deficiency alleviates homocysteine-induced aortic valve calcification.

To evaluate the role and therapeutic value of homocysteine (hcy)-inducible endoplasmic reticulum stress (ERS) protein with ubiquitin like domain 1 (Herpud1) in hcy-induced calcific aortic valve disease (CAVD). The morbidity and mortality rates of calcific aortic valve disease (CAVD) remain high while treatment options are limited. In vivo, we use the low-density lipoprotein receptor (LDLR) and Herpud1 double knockout (LDLR-/-/Herpud1-/-) mice and used high methionine diet (HMD) to assess of aortic valve calcification lesions, ERS activation, autophagy, and osteogenic differentiation of aortic valve interstitial cells (AVICs). In vitro, the role of Herpud1 in the Hcy-related osteogenic differentiation of AVICs was investigated by manipulating of Herpud1 expression. Herpud1 was highly expressed in calcified human and mouse aortic valves as well as primary aortic valve interstitial cells (AVICs). Hcy increased Herpud1 expression through the ERS pathway and promoted CAVD progression. Herpud1 deficiency inhibited hcy-induced CAVD in vitro and in vivo. Herpud1 silencing activated cell autophagy, which subsequently inhibited hcy-induced osteogenic differentiation of AVICs. ERS inhibitor 4-phenyl butyric acid (4-PBA) significantly attenuated aortic valve calcification in HMD-fed low-density lipoprotein receptor-/- (LDLR-/-) mice by suppressing ERS and subsequent Herpud1 biosynthesis. These findings identify a previously unknown mechanism of Herpud1 upregulation in Hcy-related CAVD, suggesting that Herpud1 silencing or inhibition is a viable therapeutic strategy for arresting CAVD progression. • Herpud1 is upregulated in the leaflets of Hcy-treated mice and patients with CAVD. • In mice, global knockout of Herpud1 alleviates aortic valve calcification and Herpud1 silencing activates cell autophagy, inhibiting osteogenic differentiation of AVICs induced by Hcy. • 4-PBA suppressed Herpud1 expression to alleviate AVIC calcification in Hcy treated AVICs and to mitigate aortic valve calcification in mice.

Extensive diet-induced atherosclerosis in scavenger receptor class B type 1-deficient mice is associated with substantial leukocytosis and elevated vascular cell adhesion molecule-1 expression in coronary artery endothelium.

High levels of low density lipoprotein (LDL) cholesterol and low levels of high density lipoprotein (HDL) cholesterol are risk factors for cardiovascular disease. Mice that lack genes involved in the clearance of LDL from the bloodstream, such as the LDL receptor and apolipoprotein E, are widely used models of experimental atherosclerosis. Conversely, mice that lack the HDL receptor, scavenger receptor class B type I, and therefore have disrupted HDL functionality, also develop diet-inducible atherosclerosis but are a seldom-used disease model. In this study, we compared atherosclerosis and associated phenotypes in scavenger receptor class B type I knockout mice with those of wild type, LDL receptor knockout, and apolipoprotein E knockout mice after 20weeks of being fed an atherogenic diet containing sodium cholate. We found that while scavenger receptor class B type I knockout mice had substantially lower plasma cholesterol than LDL receptor and apolipoprotein E knockout mice, they developed atherosclerotic plaques with similar sizes and compositions in their aortic sinuses, and more extensive atherosclerosis in their descending aortas and coronary arteries. This was associated with elevated tumor necrosis factor alpha levels in scavenger receptor class B type I knockout mice compared to wild type and LDL receptor knockout mice, and lymphocytosis, monocytosis, and elevated vascular cell adhesion molecule expression in coronary artery endothelial cells compared to the other mice examined. We conclude that extensive atherosclerosis in arteries that are not generally susceptible to atherosclerosis in scavenger receptor class B type I knockout mice is driven by factors in addition to hypercholesterolemia, including inflammation, dysregulation of the immune system and increased sensitivity of endothelial cells in arteries that are normally resistant to atherosclerosis. Scavenger receptor class B type I knockout mice fed a cholate containing atherogenic diet may prove to be a useful model to study mechanisms of atherosclerosis and evaluate treatments that rely on intact LDL clearance pathways.

Cholesterol accumulation in macrophages drives NETosis in atherosclerotic plaques via IL-1β secretion.

Neutrophil extracellular trap formation (NETosis) increases atherosclerotic plaque vulnerability and athero-thrombosis. However, mechanisms promoting NETosis during atherogenesis are poorly understood. We have shown that cholesterol accumulation due to myeloid cell deficiency of the cholesterol transporters ATP Binding Cassette A1 and G1 (ABCA1/G1) promotes NLRP3 inflammasome activation in macrophages and neutrophils and induces prominent NETosis in atherosclerotic plaques. We investigated whether NETosis is a cell-intrinsic effect in neutrophils or is mediated indirectly by cellular crosstalk from macrophages to neutrophils involving IL-1β. We generated mice with neutrophil or macrophage-specific Abca1/g1 deficiency (S100A8CreAbca1fl/flAbcg1fl/fl or CX3CR1CreAbca1fl/flAbcg1fl/fl mice, respectively), and transplanted their bone marrow into low-density lipoprotein receptor knockout mice. We then fed the mice a cholesterol-rich diet. Macrophage, but not neutrophil Abca1/g1 deficiency activated inflammasomes in macrophages and neutrophils, reflected by caspase-1 cleavage, and induced NETosis in plaques. NETosis was suppressed by administering an interleukin (IL)-1β neutralizing antibody. The extent of NETosis in plaques correlated strongly with the degree of neutrophil accumulation, irrespective of blood neutrophil counts, and neutrophil accumulation was decreased by IL-1β antagonism. In vitro, IL-1β or media transferred from Abca1/g1-deficient macrophages increased NETosis in both control and Abca1/Abcg1 deficient neutrophils. This cell-extrinsic effect of IL-1β on NETosis was blocked by an NLRP3 inhibitor. These studies establish a new link between inflammasome-mediated IL-1β production in macrophages and NETosis in atherosclerotic plaques. Macrophage-derived IL-1β appears to increase NETosis both by increasing neutrophil recruitment to plaques and by promoting neutrophil NLRP3 inflammasome activation.

LAG3 Regulates T Cell Activation and Plaque Infiltration in Atherosclerotic Mice

BackgroundThe immune checkpoint receptor lymphocyte-activation gene 3 (LAG3) is a new target for immune checkpoint blockade (ICB), but the effects of LAG3 on atherosclerosis are not known. ObjectivesThe aim of the study was to evaluate the role of LAG3 on plaque inflammation using murine hypercholesterolemic models of atherosclerosis. MethodsTo study the role of LAG3 in atherosclerosis, we investigated both bone marrow chimeras lacking LAG3 in hematopoietic cells as well as global Lag3-/- knockout mice. Effects of anti-LAG3 monoclonal antibody monotherapy and combination therapy with anti-programmed cell death protein 1 (PD-1) were tested in hypercholesterolemic low-density lipoprotein receptor knockout (Ldlr-/-) mice and evaluated by histology and flow cytometry. ResultsLAG3-deficiency or treatment with blocking anti-LAG3 monoclonal antibodies led to increased levels of both interferon gamma–producing T helper 1 cells and effector/memory T cells, balanced by increased levels of regulatory T cells. Plaque size was affected by neither LAG3 deficiency nor LAG3 blockade, although density of T cells in plaques was 2-fold increased by loss of LAG3. Combination therapy of anti-PD-1 and anti-LAG3 had an additive effect on T cell activation and cytokine production and promoted plaque infiltration of T cells. ConclusionsLoss of LAG3 function promoted T cell activation and accumulation in plaques while not affecting plaque burden. Our report supports further clinical studies investigating cardiovascular risk in patients treated with anti-LAG3 ICB.

Aerobic Exercise Training Reduces Atherogenesis Induced by Low-Sodium Diet in LDL Receptor Knockout Mice.

This study investigated the efficacy of aerobic exercise training (AET) in the prevention of dyslipidemia, insulin resistance (IR), and atherogenesis induced by severe low-sodium (LS) diet. LDL receptor knockout (LDLR KO) mice were fed a low-sodium (LS) (0.15% NaCl) or normal-sodium (NS; 1.27% NaCl) diet, submitted to AET in a treadmill, 5 times/week, 60 min/day, 15 m/min, for 90 days, or kept sedentary. Blood pressure (BP), plasma total cholesterol (TC) and triglyceride (TG) concentrations, lipoprotein profile, and insulin sensitivity were evaluated at the end of the AET protocol. Lipid infiltration, angiotensin II type 1 receptor (AT1), receptor for advanced glycation end products (RAGE), carboxymethyllysine (CML), and 4-hydroxynonenal (4-HNE) contents as well as gene expression were determined in the brachiocephalic trunk. BP and TC and gene expression were similar among groups. Compared to the NS diet, the LS diet increased vascular lipid infiltration, CML, RAGE, 4-HNE, plasma TG, LDL-cholesterol, and VLDL-TG. Conversely, the LS diet reduced vascular AT1 receptor, insulin sensitivity, HDL-cholesterol, and HDL-TG. AET prevented arterial lipid infiltration; increases in CML, RAGE, and 4-HNE contents; and reduced AT1 levels and improved LS-induced peripheral IR. The current study showed that AET counteracted the deleterious effects of chronic LS diet in an atherogenesis-prone model by ameliorating peripheral IR, lipid infiltration, CML, RAGE, 4-HNE, and AT1 receptor in the intima-media of the brachiocephalic trunk. These events occurred independently of the amelioration of plasma-lipid profile, which was negatively affected by the severe dietary-sodium restriction.

Low-density lipoprotein receptor deficiency reduced bone mass in mice via the c-fos/NFATc1 pathway

AimThe low-density lipoprotein receptor (LDLR) plays a crucial role in regulating lipid metabolism. However, whether LDLR deficiency affects bone mass and morphology remains controversial. This study aimed to analyze the bone phenotypes of LDLR knockout (LDLR−/−) mice. Main methodsEight-week-old LDLR−/− and wild-type (WT) mice were subjected to microcomputed tomography to detect bone phenotypes. Enzyme-linked immunosorbent assay kits were used to detect the serum estrogen levels and matrix metalloproteinase 9 (MMP-9) levels in tissue homogenates. Von Kossa, toluidine blue, tartrate-resistant acid phosphatase (TRAP) staining, and calcein labeling were performed to explore bone turnover parameters. In vitro, osteoclastogenesis was induced in bone marrow cells from LDLR−/− mice and WT mice in the presence or absence of 17β-estradiol. The microphotographs and number of osteoclasts were validated using TRAP staining. Relative gene expression during osteoclast differentiation and maturation was determined by quantitative real-time polymerase chain reaction. Key findingsLDLR deficiency results in reduced bone mineral density of the tibial cancellous bone, indicating bone loss to some extent in LDLR−/− mice. LDLR deficiency significantly increased the number of osteoclasts, but not osteoblasts. In vitro, bone marrow cells from LDLR−/− mice displayed enhanced osteoclastic potential along with increased expression of TRAP, cathepsin K, nuclear factor of activated T-cells 1 (NFATc1), c-fos, and MMP-9 and inhibited dendritic cell-specific transmembrane protein expression. Moreover, 17β-estradiol treatment can inhibit osteoclastogenesis in vitro. SignificanceOur data demonstrated that LDLR deficiency promoted osteoclastogenesis by upregulating c-fos and NFATc1 expression, reducing cancellous bone mass in LDLR−/− mice.

Identification of a PRDM1-regulated T cell network to regulate T cell driving plaque inflammation in human and mouse atherosclerosis

Abstract T cells have a prominent role in the pathogenesis of atherosclerosis, although their exact function remains elusive. Here, we pursued a network-driven approach to identify T cell-associated gene programs driving the transition from low- to high-risk human plaques. In this study 43 human carotid arterial plaques were collected and stratified based on absence (low-risk) or presence (high-risk) of intraplaque haemorrhage (IPH). Lesion RNA was subjected to microarray gene expression analysis and analysed by Weighted Gene Co-expression Network Analysis (WGCNA). We identified a co-expressed gene cluster displaying a strong T cell signalling signature in high- versus low-risk plaque, which was tightly connected to subnetworks of angiogenesis and interferon-signalling. WGCNA-based Bayesian network inference, cell-type deconvolution and single-cell gene expression revealed that this T cell-associated gene program was likely linked to effector-memory cytotoxic CD8+ T cells, underpinning the central role of T cells in plaque destabilization. Gene regulatory analysis identified cytotoxic T cell-related transcription factors, like PRDM1, regulating this plaque T cell gene program. Moreover, we demonstrated in LDL receptor knockout mice with T cell-specific Prdm1 deficiency, that lack of Prdm1 in T cells resulted in larger, more advanced plaques. In conclusion, our study reveals a PRDM1-regulated T cell footprint in high- versus low-risk human atherosclerotic lesions and murine atherosclerotic plaque development, thereby identifying this network as a potential target for intervention in adverse T cell responses. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): The European Research Area Network on Cardiovascular Diseases (ERA-CVD and Dutch Heart Foundation)

Deletion of Macrophage-Specific Glycogen Synthase Kinase (GSK)-3α Promotes Atherosclerotic Regression in Ldlr-/- Mice.

Recent evidence from our laboratory suggests that impeding ER stress–GSK3α/β signaling attenuates the progression and development of atherosclerosis in mouse model systems. The objective of this study was to determine if the tissue-specific genetic ablation of GSK3α/β could promote the regression of established atherosclerotic plaques. Five-week-old low-density lipoprotein receptor knockout (Ldlr−/−) mice were fed a high-fat diet for 16 weeks to promote atherosclerotic lesion formation. Mice were then injected with tamoxifen to induce macrophage-specific GSK3α/β deletion, and switched to standard diet for 12 weeks. All mice were sacrificed at 33 weeks of age and atherosclerosis was quantified and characterized. Female mice with induced macrophage-specific GSK3α deficiency, but not GSK3β deficiency, had reduced plaque volume (~25%) and necrosis (~40%) in the aortic sinus, compared to baseline mice. Atherosclerosis was also significantly reduced (~60%) in the descending aorta. Macrophage-specific GSK3α-deficient mice showed indications of increased plaque stability and reduced inflammation in plaques, as well as increased CCR7 and ABCA1 expression in lesional macrophages, consistent with regressive plaques. These results suggest that GSK3α ablation promotes atherosclerotic plaque regression and identify GSK3α as a potential target for the development of new therapies to treat existing atherosclerotic lesions in patients with cardiovascular disease.

Differential Effect of Dietary Supplementation with a Soybean Oil Enriched in Oleic Acid versus Linoleic Acid on Plasma Lipids and Atherosclerosis in LDLR-Deficient Mice.

Both monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) play important roles in lipid metabolism, and diets enriched with either of these two fatty acids are associated with decreased cardiovascular risk. Conventional soybean oil (CSO), a common food ingredient, predominantly contains linoleic acid (LA; C18:2), a n-6 PUFA. Recently, a modified soybean oil (MSO) enriched in oleic acid (C18:1), a n-9 MUFA, has been developed, because of its improved chemical stability to oxidation. However, the effect of the different dietary soybean oils on cardiovascular disease remains unknown. To test whether diets rich in CSO versus MSO would attenuate atherosclerosis development, LDL receptor knock-out (LDLR-KO) mice were fed a Western diet enriched in saturated fatty acids (control), or a Western diet supplemented with 5% (w/w) LA-rich CSO or high-oleic MSO for 12 weeks. Both soybean oils contained a similar amount of linolenic acid (C18:3 n-3). The CSO diet decreased plasma lipid levels and the cholesterol content of VLDL and LDL by approximately 18% (p < 0.05), likely from increased hepatic levels of PUFA, which favorably regulated genes involved in cholesterol metabolism. The MSO diet, but not the CSO diet, suppressed atherosclerotic plaque size compared to the Western control diet (Control Western diet: 6.5 ± 0.9%; CSO diet: 6.4 ± 0.7%; MSO diet: 4.0 ± 0.5%) (p < 0.05), independent of plasma lipid level changes. The MSO diet also decreased the ratio of n-6/n-3 PUFA in the liver (Control Western diet: 4.5 ± 0.2; CSO diet: 6.1 ± 0.2; MSO diet: 2.9 ± 0.2) (p < 0.05), which correlated with favorable hepatic gene expression changes in lipid metabolism and markers of systemic inflammation. In conclusion, supplementation of the Western diet with MSO, but not CSO, reduced atherosclerosis development in LDLR-KO mice independent of changes in plasma lipids.

The effect of α-linolenic acid enrichment in perinatal diets in preventing high fat diet-induced SCD1 increased activity and lipid disarray in adult offspring of low density lipoprotein receptor knockout (LDLRKO) mice

The present study examined the effects of maternal perinatal dietary ALA enrichment on the high fat diet (HFD)-induced lipid disarray in the adult offspring of low density lipoprotein receptor knock-out (LDLRKO) mice.Female LDLRKO mice received, during pregnancy and lactation, isocaloric diets with either corn oil, RD, or flax oil, ALA. The weaning offspring was given a regular chow diet for a washout period of eight weeks, which was followed by HFD for eight weeks. Plasma and liver lipids and SCD1 activity were then analyzed.The HFD-fed RD adult offspring had substantially higher plasma cholesterol levels than the HFD-fed ALA offspring (15.7 versus 9.7 mmole/l, p<0.00001) and non-alcoholic fatty liver disease (NAFLD) (65.0 versus 23.9 mg/g lipids, p<0.00001). Liver lipids oleic acid (OA) content and monounsaturated to saturated fatty acids (MUFA/SAT) ratio, were two times lower in RD compared to ALA (p<0.0001). The threefold HFD-induced SCD1 raised activity (p<0.00001), and OA produced from SA, observed in RD adult offspring were prevented by perinatal ALA.In conclusion, the resilience of SCD1 to HFD- induced increased activity may account for the beneficial effects of perinatal ALA dietary enrichment in preventing NAFLD and hypercholesterolemia from occurring in adult LDLRKO offspring mice.