Mice Fed Western Diet Research Articles

Elongation factor 1A1 inhibition elicits changes in lipid droplet size, the bulk transcriptome, and cell type-associated gene expression in MASLD mouse liver.

Eukaryotic elongation factor 1A1 (EEF1A1), originally identified for its role in protein synthesis, has additional functions in diverse cellular processes. Of note, we previously discovered a role for EEF1A1 in hepatocyte lipotoxicity. We also demonstrated that a 2-wk intervention with the EEF1A1 inhibitor didemnin B (DB) (50 µg/kg) decreased liver steatosis in a mouse model of obesity and metabolic dysfunction-associated steatotic liver disease (MASLD) [129S6/SvEvTac mice fed Western diet (42% fat) for 26 wk]. Here, we further characterized the hepatic changes occurring in these mice by assessing lipid droplet (LD) size, bulk differential expression, and cell type-associated alterations in gene expression. Consistent with the previously demonstrated decrease in hepatic steatosis, we observed decreased median LD size in response to DB. Bulk RNA sequencing (RNA-Seq) followed by gene set enrichment analysis revealed alterations in pathways related to energy metabolism and proteostasis in DB-treated mouse livers. Deconvolution of bulk data identified decreased cell type association scores for cholangiocytes, mononuclear phagocytes, and mesenchymal cells in response to DB. Overrepresentation analyses of bulk data using cell type marker gene sets further identified hepatocytes and cholangiocytes as the primary contributors to bulk differential expression in response to DB. Thus, we show that chemical inhibition of EEF1A1 decreases hepatic LD size and decreases gene expression signatures associated with several liver cell types implicated in MASLD progression. Furthermore, changes in hepatic gene expression were primarily attributable to hepatocytes and cholangiocytes. This work demonstrates that EEF1A1 inhibition may be a viable strategy to target aspects of liver biology implicated in MASLD progression.NEW & NOTEWORTHY Chemical inhibition of EEF1A1 decreases hepatic lipid droplet size and decreases gene expression signatures associated with liver cell types that contribute to MASLD progression. Furthermore, changes in hepatic gene expression are primarily attributable to hepatocytes and cholangiocytes. This work highlights the therapeutic potential of targeting EEF1A1 in the setting of MASLD, and the utility of RNA-Seq deconvolution to reveal valuable information about tissue cell type composition and cell type-associated gene expression from bulk RNA-Seq data.

Renal Proximal Tubule Cell-specific Megalin Deletion Does Not Affect Atherosclerosis But Induces Tubulointerstitial Nephritis in Mice Fed Western Diet.

Pharmacological inhibition of megalin (also known as low-density lipoprotein receptor-related protein 2: LRP2) attenuates atherosclerosis in hypercholesterolemic mice. Since megalin is abundant in renal proximal tubule cells (PTCs), the purpose of this study was to determine whether PTC-specific deletion of megalin reduces hypercholesterolemia-induced atherosclerosis in mice. Female Lrp2 f/f mice were bred with male Ndrg1-Cre ERT2 +/0 mice to develop PTC-LRP2 +/+ and -/- littermates. To study atherosclerosis, all mice were bred to an LDL receptor -/- background and fed a Western diet to induce atherosclerosis. PTC-specific megalin deletion did not attenuate atherosclerosis in LDL receptor -/- mice in either sex. Serendipitously, we discovered that PTC-specific megalin deletion led to interstitial infiltration of CD68+ cells and tubular atrophy. The pathology was only evident in male PTC-LRP2 -/- mice fed the Western diet, but not in mice fed a normal laboratory diet. Renal pathologies were also observed in male PTC-LRP2 -/- mice in an LDL receptor +/+ background fed the same Western diet, demonstrating that the renal pathologies were dependent on diet and not hypercholesterolemia. In contrast, female PTC-LRP2 -/- mice had no apparent renal pathologies. In vivo multiphoton microscopy demonstrated that PTC-specific megalin deletion dramatically diminished albumin accumulation in PTCs within 10 days of Western diet feeding. RNA sequencing analyses demonstrated the upregulation of inflammation-related pathways in kidney. PTC-specific megalin deletion does not affect atherosclerosis, but leads to tubulointerstitial nephritis in mice fed Western diet, with severe pathologies in male mice.

Effects of Age and Diet on Triglyceride Metabolism in Mice.

Both age and diet can contribute to alterations in triglyceride metabolism and subsequent metabolic disease. In humans, plasma triglyceride levels increase with age. Diets high in saturated fats can increase triglyceride levels while diets high in omega-3 fatty acids decrease triglyceride levels. Here we asked how age and long-term diet effected triglyceride metabolism in mice. We fed male and female mice a low-fat diet, a western diet, or a diet high in polyunsaturated and omega-3 (n-3) fatty acids for up to 2 years. We measured survival, body composition, plasma triglyceride levels, chylomicron clearance, and oral fat, glucose, and insulin tolerance. Triglyceride levels in mice did not increase with age, regardless of diet. Oral fat tolerance increased with age, while chylomicron clearance remained unchanged. Mice fed western diet had decreased survival. Interestingly, mice fed the n-3 diet gained more lean mass, and had lower insulin levels than mice fed either low-fat or western diet. Moreover, triglyceride uptake into the hearts of mice fed the n-3 diet was strikingly higher than in other groups. In mice, age-induced changes in triglyceride metabolism did not match those in humans. Our data suggested that mice, like humans, had decreased fat absorption with age, but plasma triglyceride clearance did not decrease with age in mice, resulting in lower plasma triglyceride levels and improved oral fat tolerance with age. A chronic diet high in n-3 fatty acids increased insulin sensitivity and uptake of triglycerides specifically into the heart but how these observations are connected is unclear. The changes in triglyceride metabolism that occur with age in humans are not reflected in a mouse model, thus mice are likely not an ideal model for understanding how age impacts lipid metabolism and subsequent metabolic disease.A fish-oil based high-fat diet high in omega-3 fatty acids significantly increases fatty acid uptake in the heart while at the same time decreases fasting insulin levels.In future studies it will be important to understand how the omega-3 fatty acid induced increase in fatty acid uptake affects cardiac function and how it is related to other phenotypes induced by omega-3 fatty acids.

Enhancement of high-density lipoprotein-associated protease inhibitor activity prevents atherosclerosis progression

Background and aimsInflammatory cells within atherosclerotic lesions secrete proteolytic enzymes that contribute to lesion progression and destabilization, increasing the risk for an acute cardiovascular event. Elastase is a serine protease, secreted by macrophages and neutrophils, that may contribute to the development of unstable plaque. We previously reported interaction of endogenous protease-inhibitor proteins with high-density lipoprotein (HDL), including alpha-1-antitrypsin, an inhibitor of elastase. These findings support a potential role for HDL as a modulator of protease activity. In this study, we test the hypothesis that enhancement of HDL-associated elastase inhibitor activity is protective against atherosclerotic lesion progression. MethodsWe designed an HDL-targeting protease inhibitor (HTPI) that binds to HDL and confers elastase inhibitor activity. Lipoprotein binding and the impact of HTPI on atherosclerosis were examined using mouse models. Histology and immunofluorescence staining of aortic root sections were used to examine the impact of HTPI on lesion morphology and inflammatory features. ResultsHTPI is a small (1.6 kDa) peptide with an elastase inhibitor domain, a soluble linker, and an HDL-targeting domain. When incubated with human plasma ex vivo, HTPI predominantly binds to HDL. Intravenous administration of HTPI to mice resulted in its binding to plasma HDL and increased elastase inhibitor activity on isolated HDL. Accumulation of HTPI within plaque was observed after administration to Apoe−/− mice. To examine the effect of HTPI treatment on atherosclerosis, prevention and progression studies were performed using Ldlr−/− mice fed Western diet. In both study designs, HTPI-treated mice had reduced lipid deposition in plaque. ConclusionsThese data support the hypothesis that HDL-associated anti-elastase activity can improve the atheroprotective potential of HDL and highlight the potential utility of HDL enrichment with anti-protease activity as an approach for stabilization of atherosclerotic lesions.

Novel Role of Endothelial CD47 in the Regulation of Pathogenesis of Coronary Artery Disease

Background: The anti-phagocytic molecule CD47 is upregulated in atherosclerotic plaques of patients with CVD. However, the role of endothelial cell-specific expression of CD47 in the pathogenesis and progression of atherosclerosis has not yet been fully defined. Kojima et.al. described atheroprotective effect of anti-CD47 antibody by enhancing efferocytosis in atherosclerosis. In this study, we determined whether endothelial cell-specific CD47 regulates efferocytosis in atherosclerosis. Methods: Endothelial cell-specific, tamoxifen-inducible CD47knockout (CD47iECKO) mice were injected with a single dose of AAV-mutant PCSK9 via the tail vein. Susceptibility to atherosclerosis was evaluated in cross sections of the aortic sinus in mice fed Western Diet for 12 weeks. Using single-cell RNA sequencing and in vitro efferocytosis assays, we investigated the role of CD47 in regulating efferocytosis in an atherosclerotic mouse model. Results: Endothelial cell-specific deletion of CD47 led to a significant plaque reduction in the aortic sinus of athero-CD47iECKO mice. Cluster analysis and gene expression profiles of 20,000 aortic endothelial cells from athero-control and athero-CD47iECKO mice identified 10 major cell types from the integrated data: endothelial cells, vascular smooth muscle cells, fibroblasts, immune cells (B cells, T cells, macrophage), erythrocytes, oligodendrocytes cells, Schwann cells, and pericytes. After Western Diet intake, in the CD47iECKO group, a subpopulation of endothelial cells as well as fibroblasts, T-cells, B-cells, resident aortic macrophages, erythrocytes, oligodendrocytes, and pericytes were increased, while vascular smooth muscle cell numbers decreased. Using an in vitro efferocytosis assay on isolated primary endothelial cells from control and CD47iECKO mice, we observed CD47 deletion induced engulfment of apoptotic Jurkat cells, which had been exposed to BAY11-7082 (NF-κB inhibitor) to stimulate the apoptotic attributes. Quantitative PCR analyses showed that CD47 deletion increases the expression of efferocytosis receptor FasL, CX3CL1, Gpr132, MerTK, Scarb1, Sirpa, TSP1, and HMGB1. Conclusion: Our study indicates that genetic deletion of CD47 in endothelial cells attenuates atheroma progression by enhancing efferocytosis. This study also reveals phenotypic heterogenicity of endothelial cells at the single cell level during atherogenesis and provides a deeper understanding of endothelial cell biology in atherosclerosis that may facilitate the development of novel therapeutic interventions for this cardiovascular disease. NIH, AHA. 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.

Mitochondrial dysfunction and metabolic reprogramming induce macrophage pro-inflammatory phenotype switch and atherosclerosis progression in aging.

Aging increases the risk of atherosclerotic vascular disease and its complications. Macrophages are pivotal in the pathogenesis of vascular aging, driving inflammation and atherosclerosis progression. NOX4 (NADPH oxidase 4) expression increases with age, correlating with mitochondrial dysfunction, inflammation, and atherosclerosis. We hypothesized that the NOX4-dependent mitochondrial oxidative stress promotes aging-associated atherosclerosis progression by causing metabolic dysfunction and inflammatory phenotype switch in macrophages. We studied atherosclerotic lesion morphology and macrophage phenotype in young (5-month-old) and aged (16-month-old) Nox4 -/-/Apoe -/- and Apoe -/- mice fed Western diet. Young Nox4-/-/Apoe-/- and Apoe-/- mice had comparable aortic and brachiocephalic artery atherosclerotic lesion cross-sectional areas. Aged mice showed significantly increased lesion area compared with young mice. Aged Nox4-/-/Apoe-/- had significantly lower lesion areas than Apoe-/- mice. Compared with Apoe-/- mice, atherosclerotic lesions in aged Nox4-/-/Apoe-/- showed reduced cellular and mitochondrial ROS and oxidative DNA damage, lower necrotic core area, higher collagen content, and decreased inflammatory cytokine expression. Immunofluorescence and flow cytometry analysis revealed that aged Apoe-/- mice had a higher percentage of classically activated pro-inflammatory macrophages (CD38+CD80+) in the lesions. Aged Nox4-/-/Apoe-/- mice had a significantly higher proportion of alternatively activated pro-resolving macrophages (EGR2+/CD163+CD206+) in the lesions, with an increased CD38+/EGR2+ cell ratio compared with Apoe-/- mice. Mitochondrial respiration assessment revealed impaired oxidative phosphorylation and increased glycolytic ATP production in macrophages from aged Apoe-/- mice. In contrast, macrophages from Nox4-/-/Apoe-/- mice were less glycolytic and more aerobic, with preserved basal and maximal respiration and mitochondrial ATP production. Macrophages from Nox4-/-/Apoe-/- mice also had lower mitochondrial ROS levels and reduced IL1β secretion; flow cytometry analysis showed fewer CD38+ cells after IFNγ+LPS treatment and more EGR2+ cells after IL4 treatment than in Apoe-/- macrophages. In aged Apoe-/- mice, inhibition of NOX4 activity using GKT137831 significantly reduced macrophage mitochondrial ROS and improved mitochondrial function, resulting in decreased CD68+CD80+ and increased CD163+CD206+ lesion macrophage proportion and attenuated atherosclerosis. Our findings suggest that increased NOX4 in aging drives macrophage mitochondrial dysfunction, glycolytic metabolic switch, and pro-inflammatory phenotype, advancing atherosclerosis. Inhibiting NOX4 or mitochondrial dysfunction could alleviate vascular inflammation and atherosclerosis, preserving plaque integrity.

Single ethanol binge causes severe liver injury in mice fed Western diet.

Alcohol-associated liver disease (ALD) and NAFLD often coexist in Western societies that consume energy-rich and cholesterol-containing Western diets. Increased rates of ALD mortality in young people in these societies are likely attributable to binge drinking. It is largely unknown how alcohol binge causes liver damage in the setting of Western diets. In this study, we showed that a single ethanol binge (5 g/kg body weight) induced severe liver injury as shown by marked increases in serum activities of the 2 aminotransferases AST and ALT in C57BL/6J mice that have been fed a Western diet for 3 weeks. The Western diet plus binge ethanol-fed mice also displayed severe lipid droplet deposition and high contents of triglycerides and cholesterol in the liver, which were associated with increased lipogenic and reduced fatty acid oxidative gene expression. These animals had the highest Cxcl1 mRNA expression and myeloperoxidase (MPO)-positive neutrophils in the liver. Their hepatic ROS and lipid peroxidation were the highest, but their hepatic levels of mitochondrial oxidative phosphorylation proteins remained largely unaltered. Hepatic levels of several ER stress markers, including mRNAs for CHOP, ERO1A, ERO1B, BIM, and BIP, as well as Xbp1 splicing and proteins for BIP/GRP78 and IRE-α were also the highest in these animals. Interestingly, Western diet feeding for 3 weeks or ethanol binge dramatically increased hepatic caspase 3 cleavage, and the combination of the 2 did not further increase it. Thus, we successfully established a murine model of acute liver injury by mimicking human diets and binge drinking. This simple Western diet plus single ethanol binge model recapitulates major hepatic phenotypes of ALD, including steatosis and steatohepatitis characterized by neutrophil infiltration, oxidative stress, and ER stress.

Abstract 104: Gene Regulation By RNA Granule Formation In Atherosclerosis

Background: Atherosclerosis, the leading cause of cardiovascular disease related death, is a chronic inflammatory condition incited by the accumulation of low-density lipoproteins (LDL) and recruitment of macrophages and smooth muscle cells (SMCs) in the arterial intima. Both macrophages and SMCs become lipid laden foam cells that persist in the arterial vasculature, inciting chronic inflammation and plaque formation. A better understanding of the mechanisms underlying this maladaptive inflammatory response may reveal novel therapeutic strategies for intervention. Hypothesis: We hypothesize that the transformation into a foam cell state, a process that requires transcriptional rewiring, is facilitated by stress granules (SGs). SGs are cytoplasmic membrane-less organelles that sequester translation initiation factors, mRNAs, and noncoding RNAs as a result of stress. We previously reported that SG formation is increased in macrophages and vascular smooth muscle cells (SMCs) in plaques of atherosclerotic mice, and can be induced in vitro during cellular cholesterol loading. However, the consequences of SG formation and the transcripts sequestered by these organelles remain unknown Methods and Results: A current limitation in studying SG biology is the lack of in vivo models to track and isolate these dynamic structures. Using CRISPR-Cas9, we generated mice with a 3x-HA-tag on the SG marker G3BP1 in C57BL6 mice, allowing us to identify and purify SGs in vivo and in isolated primary cells. In HA-tagged G3BP1 mice fed western diet, we observe staining for HA-G3BP1 in macrophages and smooth muscle cells localized to the necrotic core and fibrous cap of atherosclerotic plaque. Furthermore, qRT-PCR on lipid laden cells revealed changes in known stress granule markers and transcripts involved in immune regulation. Conclusion: Our data support a role for SGs in repressing the translation of RNA transcripts in lipid-laden foam cells that accumulate in atherosclerotic plaque. Further study of the specific proteins and RNA transcripts sequestered in SGs of foam cells may reveal how they alter gene expression to facilitate cellular phenotypic changes in the plaque.

Abstract 305: Dietary β-carotene Accelerates Atherosclerotic Resolution By Promoting Treg Expansion In The Atherosclerosis Lesion

Forkhead box P3 ( FoxP3 ) + regulatory T cells (Tregs) mediate atherosclerosis resolution and regression by promoting an anti-inflammatory environment in the lesion. FoxP3 is upregulated by retinoic acid, the active form of vitamin A, but whether vitamin A status influences atherosclerosis resolution remains unexplored. We hypothesized that β-carotene, the main vitamin A precursor in human diet, accelerates atherosclerosis resolution by upregulating FoxP3 expression to promote Treg expansion. We induced atherosclerosis in FoxP3 EGFP mice fed Western diet control (WD-Control) and injected with an anti-sense oligonucleotide targeting the LDLR (ASO-LDLR) for 16 weeks. After this period, we harvested a subset of mice as Baseline, while the remaining mice underwent atherosclerosis resolution upon the interruption of ASO-LDLR infusions to recover LDLR expression and normalize plasma lipids for a period of three weeks. During this time, mice either continued on the same WD-Control or were switched to a Western diet supplemented with 50 mg of β-carotene/kg of diet (WD- β-carotene). To examine the implication of Tregs, we injected a subset of WD-β-carotene with anti-CD25 (WD-β-carotene + anti-CD25), while WD-Control and a second subset of WD- β-carotene mice were injected with IgG control. We estimated atherosclerosis resolution by quantifying macrophage and collagen contents in the lesions present at the level of the aortic root. WD-β-carotene-fed mice injected with IgG showed a reduction in macrophage content accompanied by an increase in collagen in comparison to all the remaining groups, while WD-β-carotene + anti-CD25 lesions resembled those present in WD-Control mice. We examined Treg the relative number of CD25 + FoxP3 + and CD25 - FoxP3 + cells in the spleen, blood, and the lesion. CD25 + FoxP3 + numbers decreased in response to anti-CD25 infusions in all the tissues examined. Dietary β-carotene favored an increase of 269% and 653% in CD25 - FoxP3 + Tregs in comparison to the Baseline of WD-Control groups, respectively. These effects occurred independently of changes in plasma lipids or changes in body weight. Our data show that β-carotene supplementation results in Treg expansion and acceleration of atherosclerosis resolution by increasing Treg number.

Oxidized phospholipid oxPAPC induces a dysfunctional Th1-like phenotype in regulatory T cells

Abstract Regulatory T cells (T regs) are protective in atherosclerosis but numbers are reduced during disease progression due to cell death and loss of stability. Oxidized lipids are abundant in atherosclerotic lesions and can activate innate immune cells, but there is limited information regarding their impact on T cells. Given the T regloss observed during atherosclerosis progression and increase in oxidized lipid levels in the microenvironment, we sought to determine whether an oxidized phospholipid associated with atherosclerosis, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (oxPAPC), alters T regdifferentiation and function. In vitro skews of naïve T cells showed that oxPAPC-treated T regswere less viable with a greater proportion expressing the Th1 markers T-bet, CXCR3, and IFN-γ. Th1 and Th17 skewed cultures were unaltered by oxPAPC suggesting a T regspecific effect of oxPAPC. OxPAPC-treated T regswere less suppressive in vitro and were insufficient to inhibit atherosclerosis progression in an adoptive transfer in Ldlr −/−mice fed Western diet for up to 16 weeks. A role for IFN-γ signaling in T regdysregulation was examined using T regspecific IFNγR1 deficient mice. IFNγR1 deficient CD4 +T cells skewed in the presence of oxPAPC did not adopt the Th1-like T regphenotype compared to control oxPAPC-treated T regs, indicating that IFNγR signaling was important for oxPAPC-mediated T regdysregulation. Bone marrow transplant of Ldlr −/−showed that T regIFNγR1 signaling was atherogenic in males but protective in females. Overall, our findings demonstrate that oxidized phospholipids directly impact T regdifferentiation and decrease their atheroprotective potential.

Fludrocortisone Induces Aortic Pathologies in Mice.

Background and Objective: In an experiment designed to explore the mechanisms of fludrocortisone-induced high blood pressure, we serendipitously observed aortic aneurysms in mice infused with fludrocortisone. The purpose of this study was to investigate whether fludrocortisone induces aortic pathologies in both normocholesterolemic and hypercholesterolemic mice. Methods and Results: Male adult C57BL/6J mice were infused with either vehicle (85% polyethylene glycol 400 (PEG-400) and 15% dimethyl sulfoxide (DMSO); n = 5) or fludrocortisone (12 mg/kg/day dissolved in 85% PEG-400 and 15% DMSO; n = 15) for 28 days. Fludrocortisone-infused mice had higher systolic blood pressure, compared to mice infused with vehicle. Fludrocortisone induced aortic pathologies in 4 of 15 mice with 3 having pathologies in the ascending and aortic arch regions and 1 having pathology in both the ascending and descending thoracic aorta. No pathologies were noted in abdominal aortas. Subsequently, we infused either vehicle (n = 5/group) or fludrocortisone (n = 15/group) into male ApoE −/− mice fed a normal laboratory diet or LDL receptor −/− mice fed either normal or Western diet. Fludrocortisone increased systolic blood pressure, irrespective of mouse strain or diet. In ApoE −/− mice infused with fludrocortisone, 2 of 15 mice had ascending aortic pathologies, but no mice had abdominal aortic pathologies. In LDL receptor −/− mice fed normal diet, 5 had ascending/arch pathologies and 1 had pathologies in the ascending, arch, and suprarenal aortic regions. In LDL receptor −/− mice fed Western diet, 2 died of aortic rupture in either the descending thoracic or abdominal region, and 2 of the 13 survived mice had ascending/arch aortic pathologies. Aortic pathologies included hemorrhage, wall thickening or thinning, or dilation. Only ascending aortic diameter in LDLR −/− mice fed Western diet reached statistical significance, compared to their vehicle. Conclusion: Fludrocortisone induces aortic pathologies independent of hypercholesterolemia. As indicated by the findings in mouse studies, people who are taking or have taken fludrocortisone might have an increased risk of aortic pathologies.

Abstract 560: Fludrocortisone Induces Aortic Pathologies In Mice

Objective: Fludrocortisone, a corticosteroid, is used to treat adrenocortical insufficiency. In a previous study to determine associations between fludrocortisone-induced high blood pressure and angiotensin regulation, we observed aortic aneurysms in some fludrocortisone-infused mice. The purpose of this study was to investigate fludrocortisone-induced aortic pathologies in normocholesterolemic and hypercholesterolemic mice. Methods and Results: To determine whether fludrocortisone induces aortic aneurysms in normocholesterolemic mice, male C57BL/6J at 8 - 9 weeks of age were infused with either vehicle (85% polyethylene glycol 400 (PEG-400) and 15% dimethyl sulfoxide (DMSO); N=5) or fludrocortisone (12 mg/kg/day dissolved in 85% PEG-400 and 15% DMSO; N=15) for 28 days. Fludrocortisone-infused mice had higher systolic blood pressure, compare to mice infused with vehicle. Fludrocortisone induced aortic pathologies in 4 of 15 mice with 3 having pathologies in the ascending/arch region and 1 having pathology in both the ascending and descending thoracic aorta. No pathologies were noted in abdominal aortas. To determine whether hypercholesterolemia augments fludrocortisone-induced aortic pathologies, we infused either vehicle (N=5/group) or fludrocortisone (N=15/group) into male ApoE -/- mice fed a normal diet or LDL receptor -/- mice fed either normal or Western diet. Fludrocortisone increased systolic blood pressure, irrespective of mouse strain or diet. In ApoE -/- mice infused with fludrocortisone, 2 of 15 had ascending aortic pathologies, but no mice had abdominal aortic pathologies. In LDL receptor -/- mice fed normal diet, 4 had ascending/arch aortic pathologies, 1 had pathologies in the ascending, arch and abdominal regions. In LDL receptor -/- mice fed Western diet, 2 died of aortic rupture, 4 had ascending/arch aortic pathologies, and 1 had pathologies in the ascending, arch and suprarenal aortic regions. Aortic pathologies were hemorrhage, wall thickening or thinning, or dilation. Given the low incidence, no quantification of aortic pathologies reached statistical significance. Conclusion: Fludrocortisone induces aortic pathologies with low incidence in both normo- and hypercholesterolemic mice.

Montmorency Tart Cherry Supplementation Has Modest Effects on the Gut Microbiome and Markers of Gut Integrity and Insulin Resistance in Mice Fed Western Diet

Abstract Objectives This study investigated the dose-dependent effects of freeze-dried Montmorency tart cherry (TC) supplementation on gut health and metabolic parameters in mice fed a western diet (WD). Methods Six-week-old male C57BL/6 mice were randomly assigned to dietary treatment groups in a 2 × 3 factorial design with diet (control [AIN-93M] or WD, 45% fat kcal and 26% sucrose kcal) and TC (0, 5, 10% wt/wt) as factors for 12 wks. At the end of dietary treatment, body composition was assessed by dual energy xray absorptiometry, and tissues were collected to evaluate metabolic parameters and markers of gut health. Cecal content was used for bacterial and short chain fatty acid analyses (SCFAs). Results TC at the 10% dose significantly increased the abundance of the beneficial bacterial phylum, Actinobacteria, relative to the unsupplemented groups (P = 0.018 and 0.010 vs control and WD, respectively). Relative cecal weight (P = 0.007) and SCFAs were significantly increased (P < 0.05) with TC supplementation (∼20% and 2-fold for relative cecal weight and SCFAs, respectively). Histological evaluation revealed reduced ileal villi height (P = 0.0348), width (P = 0.0042) and area (P = 0.0132) with WD, and TC did not alter this response. Overall, the expression of genes related to gut health (i.e barrier integrity marker, mucus layer formation, and inflammatory marker), were unaffected by both WD and TC supplementation. Body weight (P = 0.0012), fat mass (P = 0.007), fasting blood glucose (P = 0.001), serum total cholesterol (P < 0.0001), triglyceride (P = 0.002), leptin (P = 0.0011), plasminogen activator inhibitor 1 (P = 0.0344), and resistin (P = 0.0012) were increased with WD, and TC had no effect on these parameters. Despite modest effects on metabolic parameters, the homeostatic model assessment of insulin resistance, HOMA-IR, a commonly used tool for assessing insulin resistance, was improved by 50% with the 5% TC (P = 0.0003). Conclusions TC supplementation restored some beneficial bacteria and increased SCFAs altered by WD. However, these changes in the gut did not translate to improvement in metabolic outcomes except for HOMA-IR. The mechanism by which TC improves HOMA-IR needs to be investigated in future studies. Funding Sources Cherry Marketing Institute and the Jim and Lynn Williams Professorship.

Loss of 15-lipoxygenase disrupts Treg differentiation altering their pro-resolving functions

Regulatory T-cells (Tregs) are central in the maintenance of homeostasis and resolution of inflammation. However, the mechanisms that govern their differentiation and function are not completely understood. Herein, we demonstrate a central role for the lipid mediator biosynthetic enzyme 15-lipoxygenase (ALOX15) in regulating key aspects of Treg biology. Pharmacological inhibition or genetic deletion of ALOX15 in Tregs decreased FOXP3 expression, altered Treg transcriptional profile and shifted their metabolism. This was linked with an impaired ability of Alox15-deficient cells to exert their pro-resolving actions, including a decrease in their ability to upregulate macrophage efferocytosis and a downregulation of interferon gamma expression in Th1 cells. Incubation of Tregs with the ALOX15-derived specilized pro-resolving mediators (SPM)s Resolvin (Rv)D3 and RvD5n-3 DPA rescued FOXP3 expression in cells where ALOX15 activity was inhibited. In vivo, deletion of Alox15 led to increased vascular lipid load and expansion of Th1 cells in mice fed western diet, a phenomenon that was reversed when Alox15-deficient mice were reconstituted with wild type Tregs. Taken together these findings demonstrate a central role of pro-resolving lipid mediators in governing the differentiation of naive T-cells to Tregs.

Carbon tetrachloride (CCl4) accelerated development of non-alcoholic fatty liver disease (NAFLD)/steatohepatitis (NASH) in MS-NASH mice fed western diet supplemented with fructose (WDF)

BackgroundMultiple murine models of nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) have been established by using obesogenic diets and/or chemical induction. MS-NASH mouse (formally FATZO) is a spontaneously developed dysmetabolic strain that can progress from hepatosteatosis to moderate fibrosis when fed a western diet supplemented with 5% fructose (WDF). This study aimed to use carbon tetrachloride (CCl4) to accelerate and aggravate progression of NAFLD/NASH in MS-NASH mouse.MethodsMale MS-NASH mice at 8 weeks of age were fed WDF for the entire study. Starting at 16 weeks of age, CCl4 was intraperitoneally administered twice weekly at a dose of 0.2 mL/kg for 3 weeks or 0.08 mL/kg for 8 weeks. Obeticholic acid (OCA, 30 mg/kg, QD) was administered in both MS-NASH and C57Bl/6 mice fed WDF and treated with CCl4 (0.08 mL/kg).ResultsWDF enhanced obesity and hepatosteatosis, as well as induced moderate fibrosis in MS-NASH mice similar to previous reports. Administration of CCl4 accelerated liver fibrosis with increased bridging and liver hydroxyproline contents, but had no significant impact on liver steatosis and lipid contents. High dose CCl4 caused high mortality and dramatic elevation of ALT and ASL, while low dose CCl4 resulted in a moderate elevation of ALT and AST with low mortality. Compared to C57BI/6 mice with WDF and CCl4 (0.08 mL/kg), MS-NASH mice had more prominent hepatosteatosis and fibrosis. OCA treatment significantly lowered liver triglycerides, steatosis and fibrosis in both MS-NASH and C57Bl/6 mice fed WDF with CCl4 treatment.ConclusionsCCl4 reduced induction time and exacerbated liver fibrosis in MS-NASH mice on WDF, proving a superior NASH model with more prominent liver pathology, which has been used favorably in pharmaceutical industry for testing novel NASH therapeutics.

Western-type diet influences mortality from necrotising pancreatitis and demonstrates a central role for butyrate

ObjectiveThe gut microbiota are the main source of infections in necrotising pancreatitis. We investigated the effect of disruption of the intestinal microbiota by a Western-type diet on mortality and bacterial...

Nanoparticle-based “Two-pronged” approach to regress atherosclerosis by simultaneous modulation of cholesterol influx and efflux

Reduction of lipoprotein uptake by macrophages and stimulation of cholesterol efflux are two essential steps required for atherosclerotic plaque regression. We used the optimized mannose-functionalized dendrimeric nanoparticle (mDNP)-based platform for macrophage-specific delivery of therapeutics to simultaneously deliver SR-A siRNA (to reduce LDL uptake) and LXR ligand (LXR-L, to stimulate cholesterol efflux) – a novel “Two-pronged” approach to facilitate plaque regression. mDNP-mediated delivery of SR-A siRNA led to a significant reduction in SR-A expression with a corresponding decrease in uptake of oxLDL. Delivery of LXR-L increased expression of ABCA1/G1 and cholesterol efflux. Combined delivery of siRNA and LXR-L led to a significantly greater decrease in macrophage cholesterol content compared to either treatment alone. Administration of this in vitro optimized formulation of mDNP complexed with SR-A-siRNA and LXR-L (Two-pronged complex) to atherosclerotic LDLR−/− mice fed western diet (TD88137) led to significant regression of atherosclerotic plaques with a corresponding decrease in aortic cholesterol content.

Corrigendum: Progression and Regression of Hepatic Lesions in a Mouse Model of NASH Induced by Dietary Intervention and Its Implications in Pharmacotherapy

[This corrects the article DOI: 10.3389/fphar.2018.00410.].

Dipeptidyl peptidase-4 inhibition prevents nonalcoholic steatohepatitis\u2013associated liver fibrosis and tumor development in mice independently of its anti-diabetic effects

Nonalcoholic steatohepatitis (NASH) is a hepatic phenotype of the metabolic syndrome, and increases the risk of cirrhosis and hepatocellular carcinoma (HCC). Although increasing evidence points to the therapeutic implications of certain types of anti-diabetic agents in NASH, it remains to be elucidated whether their effects on NASH are independent of their effects on diabetes. Genetically obese melanocortin 4 receptor–deficient (MC4R-KO) mice fed Western diet are a murine model that sequentially develops hepatic steatosis, NASH, and HCC in the presence of obesity and insulin resistance. In this study, we investigated the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor anagliptin on NASH and HCC development in MC4R-KO mice. Anagliptin treatment effectively prevented inflammation, fibrosis, and carcinogenesis in the liver of MC4R-KO mice. Interestingly, anagliptin only marginally affected body weight, systemic glucose and lipid metabolism, and hepatic steatosis. Histological data and gene expression analysis suggest that anagliptin treatment targets macrophage activation in the liver during the progression from simple steatosis to NASH. As a molecular mechanism underlying anagliptin action, we showed that glucagon-like peptide-1 suppressed proinflammatory and profibrotic phenotypes of macrophages in vitro. This study highlights the glucose metabolism–independent effects of anagliptin on NASH and HCC development.

418 Ultrasound molecular imaging of the role of von willebrand factor-mediated platelet adhesion in atherogenesis

Abstract Funding Acknowledgements JSPS Overseas Research Fellowship Background Platelets are known to be both pro-inflammatory and pro-mitogenic. However, the role of platelet-endothelial interactions in the initiation and growth of atherosclerotic lesions is not well understood. Purpose We used contrast-enhanced ultrasound (CEU) molecular imaging of the arterial endothelium to test the hypothesis that platelet attachment to endothelial Von Willebrand Factor (VWF) promotes atherogenesis. Methods We studied wild-type mice (WT), low-density lipoprotein deficient mice fed western diet to produce atherosclerosis (LDLR-/-), and LDLR-/- mice also deficient for ADAMTS-13 (LDLR-/-ADAMTS13-/-) which is the enzyme responsible for proteolytic cleavage of endothelial-associated VWF. Mice were studied at 20 weeks and 30 weeks of age. A subset of LDLR-/- mice were treated with recombinant ADAMTS13 1 hr prior to study. Proximal aortic CEU molecular imaging of P-selectin, vascular cell adhesion molecule (VCAM)-1, von Willebrand factor (VWF), and platelet GPIbα was performed. Aortic distensibility was assessed using high-frequency (30 MHz) transthoracic echocardiography and tail cuff blood pressure systems. NF-κB of aorta was assessed by ELISA kit. Plaque size and composition were assessed by histology. Platelets and macrophage immunohistochemistry were also performed on confocal microscopy. Results Aortic molecular imaging signal for P-selectin, VCAM-1, VWF, and platelet adhesion was significantly higher in LDLR-/- than WT mice, and increased by 2-fold between 20 and 30 wks of age. Signal for VWF and platelet adhesion was abolished 1 h after administration of ADAMTS13, confirming that platelet adhesion was VWF-mediated. At 20 and 30 wks of age, molecular imaging signal for all targets was 2-fold higher (p < 0.01) in LDLR-/-ADAMTS13-/- versus LDLR-/- mice. The LDLR-/-ADAMTS13-/- mice also had lower aortic distensibility (p < 0.05), had a 2-fold higher NF-κB signal (p < 0.05), and had a 2-fold greater total plaque area (p < 0.01). Fluorescent immunohistochemistry confirmed that the LDLR-/-ADAMTS13-/- mice also had greater platelets (p < 0.05) and increased macrophage content (p < 0.05) than LDLR-/- mice in aortic plaque. Conclusion In early to mid-stage atherosclerosis, abnormal regulation of endothelial-associated VWF results in platelet adhesion and secondary up-regulation of endothelial inflammatory adhesion molecules, thereby promoting atherosclerotic plaque progression. These results indicate an important role of platelet-endothelial interactions in early atherogenesis. Abstract 418 Figure