Low-density Lipoprotein Receptor Knockout Research Articles

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.

ILRUN Promotes Atherosclerosis Through Lipid-Dependent and Lipid-Independent Factors.

Common genetic variation in close proximity to the ILRUN gene are significantly associated with coronary artery disease as well as with plasma lipid traits. We recently demonstrated that hepatic inflammation and lipid regulator with ubiquitin-associated domain-like and NBR1-like domains (ILRUN) regulates lipoprotein metabolism in vivo in mice. However, whether ILRUN, which is expressed in vascular cells, directly impacts atherogenesis remains unclear. We sought to determine the role of ILRUN in atherosclerosis development in mice. For our study, we generated global Ilrun-deficient (IlrunKO) male and female mice on 2 hyperlipidemic backgrounds: low density lipoprotein receptor knockout (LdlrKO) and apolipoprotein E knockout (ApoeKO; double knockout [DKO]). Compared with littermate control mice (single LdlrKO or ApoeKO), deletion of Ilrun in DKO mice resulted in significantly attenuated both early and advanced atherosclerotic lesion development, as well as reduced necrotic area. DKO mice also had significantly decreased plasma cholesterol levels, primarily attributable to non-HDL (high-density lipoprotein) cholesterol. Hepatic-specific reconstitution of ILRUN in DKO mice on the ApoeKO background normalized plasma lipids, but atherosclerotic lesion area and necrotic area remained reduced in DKO mice. Further analysis showed that loss of Ilrun increased efferocytosis receptor MerTK expression in macrophages, enhanced in vitro efferocytosis, and significantly improved in situ efferocytosis in advanced lesions. Our results support ILRUN as an important novel regulator of atherogenesis that promotes lesion progression and necrosis. It influences atherosclerosis through both plasma lipid-dependent and lipid-independent mechanisms. These findings support ILRUN as the likely causal gene responsible for genetic association of variants with coronary artery disease at this locus and suggest that suppression of ILRUN activity might be expected to reduce atherosclerosis.

The role of hepatic Surf4 in lipoprotein metabolism and the development of atherosclerosis in apoE−/− mice

Elevated plasma levels of low-density lipoprotein-C (LDL-C) increase the risk of atherosclerotic cardiovascular disease. Circulating LDL is derived from very low-density lipoprotein (VLDL) metabolism and cleared by LDL receptor (LDLR). We have previously demonstrated that cargo receptor Surfeit 4 (Surf4) mediates VLDL secretion. Inhibition of hepatic Surf4 impairs VLDL secretion, significantly reduces plasma LDL-C levels, and markedly mitigates the development of atherosclerosis in LDLR knockout (Ldlr−/−) mice. Here, we investigated the role of Surf4 in lipoprotein metabolism and the development of atherosclerosis in another commonly used mouse model of atherosclerosis, apolipoprotein E knockout (apoE−/−) mice. Adeno-associated viral shRNA was used to silence Surf4 expression mainly in the liver of apoE−/− mice. In apoE−/− mice fed a regular chow diet, knockdown of Surf4 expression significantly reduced triglyceride secretion and plasma levels of non-HDL cholesterol and triglycerides without causing hepatic lipid accumulation or liver damage. When Surf4 was knocked down in apoE−/− mice fed the Western-type diet, we observed a significant reduction in plasma levels of non-HDL cholesterol, but not triglycerides. Knockdown of Surf4 did not increase hepatic cholesterol and triglyceride levels or cause liver damage, but significantly diminished atherosclerosis lesions. Therefore, our findings indicate the potential of hepatic Surf4 inhibition as a novel therapeutic strategy to reduce the risk of atherosclerotic cardiovascular disease.

Sugar Kelp Consumption Did Not Alter Circulating and Hepatic Lipids in Low-Density Lipoprotein Receptor Knockout Mice Fed a Diet High in Fat and Cholesterol

ObjectivesWe have previously shown that sugar kelp (Saccharina latissima), a brown seaweed, decreased serum triglycerides (TG) and total cholesterol (TC), and prevented adipose tissue inflammation and fibrosis in male diet-induced obesity mice. In this study, we investigated whether the consumption of sugar kelp can prevent metabolic and inflammatory features in low-density lipoprotein receptor knockout (Ldlr KO) mice fed a diet high in fat and cholesterol. MethodsMale Ldlr KO mice at the age of 7 weeks from the Jackson laboratory were randomly assigned to a high-fat/high-cholesterol control (HF/HC; 16% fat, 0.25% cholesterol, w/w, n = 12), or a HF/HC supplemented with sugar kelp (HF/HC-SK; 6.0% dried sugar kelp power, w/w, n = 12). Mice were fed the experimental diets for 16 weeks and then serum and tissue samples were collected to measure blood and liver lipids, serum alanine aminotransferase, and hepatic gene expression. ResultsMice fed HF/HC-SK diet showed a trend towards an increase in final body weight compared to HF/HC control. Serum TG and TC concentrations were not significantly different between control and sugar kelp-fed mice. In the liver, sugar kelp consumption did not alter weights, TG, TC, and lipogenic gene expression. However, the expression of macrophage markers, such as adhesion G protein-coupled receptor E1 and cluster of differentiation 68, and M1 macrophage markers, including tumor necrosis factor, and integrin subunit alpha X, were significantly higher in HF/HC-SK group than HF/HC control mice. Despite the increases in inflammatory markers in the liver of the HF/HC-SK group, serum ALT activity was not different between the two groups. ConclusionsSugar kelp consumption did not alter circulating and hepatic TC and TG, and serum liver injury marker in HF/HC-fed Ldlr KO mice. Further study is warranted to determine whether sugar kelp can impact the development of atherosclerosis in this mouse model. Funding SourcesUSDA Hatch CONS00993.

Idol Depletion Protects against Spontaneous Atherosclerosis in a Hamster Model of Familial Hypercholesterolemia.

Inducible degrader of low-density lipoprotein (LDL) receptor (Idol) is an E3 ubiquitin ligase coded by Idol, the target gene of liver X receptor (LXR), which primarily mediates the ubiquitination and lysosomal degradation of low-density lipoprotein receptor (LDLR). Previous studies from independent groups have shown that plasma cholesterol regulation by the LXR-Idol-LDLR axis is tissue- and species-specific, indicating that the precise molecular mechanism by which Idol modulates lipid metabolism has not been completely understood and needs to be further validated in other species. Hamster, a small rodent animal model expressing endogenous cholesterol ester transfer protein (CETP), possesses many metabolic characteristics that are different from mouse but similar to human. In this study, an Idol knockout (Idol−/−) hamster model was developed using CRISPR/Cas9 gene editing system to investigate the effect of Idol depletion on plasma lipid metabolism and atherosclerosis. Our results showed that there were no significant differences in hepatic LDLR protein and plasma cholesterol levels in Idol−/− hamsters compared with wild-type (WT) controls, which was consistent with the observation that LXR agonist treatment increased the expression of Idol mRNA in the small intestine but not in the liver of WT hamsters. However, we found that plasma triglyceride (TG) levels were significantly reduced in Idol−/− hamsters due to an enhancement of TG clearance. In addition, the morphological data demonstrated that inactivation of Idol significantly lowered plasma total cholesterol and TG levels and protected against spontaneous atherosclerotic lesions in aged LDLR knockout hamsters on a chow diet but had no effect on diet-induced atherosclerosis in hamsters lacking one copy of the Ldlr gene. In conclusion, our findings suggest that Idol can regulate plasma lipid metabolism and atherosclerosis independent of LDLR function.

The Impact of the CTHRSSVVC Peptide Upon Experimental Models of Trypanosoma cruzi Infection.

Chagas disease (CD), caused by the hemoflagellate protozoan Trypanosoma cruzi, affects more than six million people worldwide and presents an unsatisfactory therapy, based on two nitroderivatives, introduced in clinical medicine for decades. The synthetic peptide, with CTHRSSVVC sequence (PepA), mimics the CD163 and TNF-α tripeptide “RSS” motif and binds to atheromatous plaques in carotid biopsies of human patients, spleen tissues, and a low-density lipoprotein receptor knockout (LDLr−/−) mouse model of atherosclerosis. CD163 receptor is present on monocytes, macrophages, and neutrophils, acting as a regulator of acute-phase processes and modulating aspects of the inflammatory response and the establishment of infections. Due to the potential theranostic role of PepA, our aim was to investigate its effect upon T. cruzi infection in vitro and in vivo. PepA and two other peptides with shuffled sequences were assayed upon different binomials of host cell/parasite, including professional [as peritoneal mouse macrophages (PMM)] and non-professional phagocytes [primary cultures of cardiac cells (CM)], under different protocols. Also, their impact was further addressed in vivo using a mouse model of acute experimental Chagas disease. Our in-vitro findings demonstrate that PepA and PepB (the peptide with random sequence retaining the “RS” sequence) reduced the intracellular parasitism of the PMM but were inactive during the infection of cardiac cells. Another set of in-vitro and in-vivo studies showed that they do not display a trypanocidal effect on bloodstream trypomastigotes nor exhibit in-vivo efficacy when administered after the parasite inoculation. Our data report the in-vitro activity of PepA and PepB upon the infection of PMM by T. cruzi, possibly triggering the microbicidal arsenal of the host professional phagocytes, capable of controlling parasitic invasion and proliferation.

Abstract 225: Lack Of Map Kinase Phosphatase-5 In Macrophages Protects Ldlr-null Mice Against Atherogenesis

Objective: Mitogen-activated protein kinases (MAPKs), including JNK, ERK, and p38 MAPK, are critical in regulating the expression of various pro-inflammatory and anti-inflammatory cytokines and chemokines. Previous work has suggested that the absence of MAP Kinase Phosphatase-5 (MKP-5) inhibits oxidized low-density lipoprotein (oxLDL)-induced macrophage foam cell formation without influencing the MAKP activation. The current study is to determine the role of macrophage MKP-5 in the pathogenesis of atherosclerosis and underlying mechanisms. Approach and Results: Nine-week-old male congenic MKP-5 deficient (MKP-5 -/- ) and C57Bl/6J control (WT) mice on an LDL receptor knock-out (LDLR -/- ) background were fed with a high-fat diet containing 1.25% cholesterol (HFD) for 14 weeks. Global deficiency of MKP-5 attenuated atherosclerotic plaque formation without altering the lipid profile in vivo. To further elucidate the macrophage-specific effect of MKP-5 in atherogenesis, Lethally irradiated LDLR -/- mice were transplanted with WT or MKP-5 -/- bone marrow and subjected to high-fat feeding. Mice transplanted with MKP-5 -/- bone marrow developed smaller atherosclerotic lesions accompanied by decreased lipid deposition and macrophage content compared to WT. Lack of MKP-5 in macrophages reduced plasma levels of interleukin-1α (IL-1α) and IL-7, elevated anti-inflammatory cytokines IL-1 receptor antagonist (IL-1rn), and IL-4. Mechanistically, lack of MKP-5 in macrophages inhibited ox-LDL-induced foam cell formation through enhanced cholesterol efflux mediated by increased expression of ATP-binding cassette transporters ABCA1 and ABCG1. Conclusions: These data suggest that the myeloid MKP-5 deficiency reduces atherosclerosis progression and foam cell formation by ameliorating cholesterol efflux and inhibiting inflammation.

Abstract 245: Antibody Responses To ApoA-I Rely On An Interplay Between B And T Cells

Apolipoprotein A-I (ApoA-I), the major protein of high-density lipoprotein (HDL), has been investigated as the target and regulatory element of immune responses in both patients and mice susceptible to atherosclerosis. Previous work by our lab has focused on investigating the immune responses to ApoA-I in wild-type mice as a strategy to understand their role in models of atherosclerosis and other inflammatory conditions. We hypothesize that peripheral immunologic tolerance is responsible for regulating the dynamic immune response to ApoA-I. To examine this, we first evaluated antibody profiles to ApoA-I in both wild-type C57BL/6 and LDL receptor knockout mice and found that anti-ApoA-I antibodies naturally develop to the protein. To determine whether this response was T cell dependent or independent, we examined whether nu/nu mice on a BALB/c background, and the parent strain, displayed the phenotype. These studies showed that both nu/nu, or wild type BALB/c mice fail to mount antibody responses to ApoA-I suggesting a regulatory mechanism might be responsible for suppressing this antibody response. Ex vivo stimulation of immune cells with ApoA-I provide evidence of both B cell and T cell activation, suggesting that peripheral tolerance mechanisms may be responsible for regulating the humoral and cellular immune response to ApoA-I. These findings agree with previous studies where ApoA-I has been shown to regulate T cell activity in the context of atherosclerosis by inducing the development of regulatory T cells, which assist in suppressing the development of plaque formation; as well as studies showing a higher propensity toward Treg development in BALB/c mice. Ongoing experiments, which more clearly characterize the role of B cells and T cells in response to ApoA-I, will be presented with the goal of clarifying the immune mechanisms responsible for anti-ApoA-I autoimmunity. These data will help guide continued understanding of the role of immune responses to ApoA-I in atherosclerotic disease progression.

Abstract 497: IL-19 Induces Lymphangiogenesis Through A Prox1-dependent Mechanism

Introduction: The role of lymphangiogenesis in atherogenesis is currently under-characterized and controversial. It has been suggested that lymphangiogenesis is either protective by promoting plaque regression through reverse cholesterol transport (RCT) or deleterious by promoting plaque instability through white blood cell trafficking. We have previously reported that Interleukin-19 (IL-19), an anti-inflammatory cytokine, uniquely attenuates atherosclerotic plaque progression while also being pro-angiogenic. This drives our hypothesis that one mechanism whereby IL-19 is atheroprotective is by promoting lymphangiogenesis. Methods &amp; Results: IL-19 can significantly induce primary human lymphatic endothelial cell (LEC) proliferation, migration, and tube formation, all angiogenic assays, to the same degree as VEGF-C, a known mediator of lymphangiogenesis. RNA sequencing (RNAseq) analysis indicates that IL-19 induces expression of chemokines as well as several angiogenic transcription factors in LECs. Notably, it induces expression of Prox1, a master transcription factor of lymphangiogenesis, 6.2-fold. The increases seen in chemokine and transcription factor expression from RNAseq were verified through qPCR analysis. Subsequent Prox1 siRNA knockdown prevented IL-19 associated LEC proliferation and migration. We then utilized an LDL Receptor Knockout mouse model fed a high fat diet for 16-weeks with daily injections of either recombinant mouse IL-19 or saline. Preliminary results suggest an increase in lymphatic vessel presence is associated with the known decrease IL-19 causes in atherosclerotic plaque progression. Transendothelial resistance assays were performed to determine IL-19's effect on lymphatic cell permeability. Preliminary data suggests that IL-19 alters LEC resistance, indicative of permeability and possibly RCT. We will also continue to evaluate RCT and inflammatory cell trafficking via intravital microscopy. Conclusion: These data suggest that IL-19 increases lymphatic vessel formation, likely through a Prox1-dependent mechanism. These results have the potential to shift our understanding toward a positive view of lymphangiogenesis and attenuation of atherosclerosis.

IL‐19 Regulates Atherosclerotic Plaque Progression via Lymphangiogenesis

IntroductionCardiovascular diseases are the leading cause of deaths globally, representing approximately 31% of deaths per year, 85% of which are due to myocardial infarction (MI) or stroke. This does not account for the millions of patients suffering daily with current therapies that are not efficacious in treating the root cause of MI and stroke, atherosclerosis, by reducing inflammation or increasing reverse cholesterol transport (RCT). Atherosclerosis is a vascular inflammatory disease characterized by the formation of fatty plaques which can rupture, leading vessel occlusion. Lymphangiogenesis is the process whereby lymphatic vessels are generated, but its role in atherogenesis is currently under‐characterized and controversial. It has been suggested that lymphangiogenesis is either protective by promoting plaque regression through RCT or it is deleterious by promoting plaque instability through white blood cell trafficking. Our lab has previously reported that Interleukin‐19 (IL‐19), an anti‐inflammatory cytokine, uniquely attenuates atherosclerotic plaque progression while also being pro‐angiogenic. This drives our hypothesis that one mechanism whereby IL‐19 is atheroprotective is by driving lymphangiogenesis, allowing attenuation of plaque progression.Methods &amp; ResultsSeveral approaches were performed to test the hypothesis that IL‐19 is lymphangiogenic. IL‐19 can significantly induce primary human lymphatic endothelial cell (LEC) proliferation, migration, and tube formation, all angiogenic assays, to the same degree as VEGF‐C, a known mediator of lymphangiogenesis. RNA sequencing (RNAseq) analysis indicates that IL‐19 induces chemokine expression as well as an angiogenic transcriptional program in LECs. Notably it induces expression of Prox1, a master transcription factor of lymphangiogenesis, 6.2‐fold. The increases of chemokine and transcription factor expression from RNAseq were verified through qPCR analysis. To further analyze the translational effects of IL‐19, we utilized an LDL Receptor Knockout (LDLR‐/‐) mouse model fed a high fat diet for 16‐weeks with daily injections of either recombinant mouse IL‐19 or saline. Preliminary results suggest an increase in lymphatic vessel presence associated with the known decrease IL‐19 causes in atherosclerotic plaque progression.Conclusion &amp; SummaryThese data suggest that IL‐19 increases lymphatic vessel formation, potentially allowing RCT to take place to decrease plaque burden in atherosclerosis. Further studies will confirm IL‐19’s mechanistic pathway in LECs. We will also determine RCT and inflammatory cell trafficking by intravital microscopy. These results have the potential to shift our understanding toward a positive view of the association between the lymphatic system and atherosclerosis.

Abstract 104: R952Q Mutation In ApoE Receptor-2 Exacerbates Hypercholesterolemia And Accelerates Atherosclerosis Progression In Ldl Receptor-knockout Mice

Background and objective: Apolipoprotein E Receptor-2 (apoER2) is a type I transmembrane receptor in the LDL receptor family and is expressed in vascular smooth muscle cells (VSMC), endothelium, and macrophages. An apoER2 mutation referred to as ApoER2-R952Q (R952Q) is associated with elevated plasma triglyceride levels and increased risk of premature coronary artery disease and myocardial infarction. Disease phenotypes emerging from the loss of apoER2 have been established, with results showing that apoER2 inactivation accelerates atherosclerosis progression through macrophage-mediated lipid accumulation and VSMC senescence to accelerate necrosis. This study investigated the effects of the R952Q mutation and its influence on atherosclerosis. Approach and results: We generated R952Q-expressing mice using a CRISPR-directed approach. This mouse line was crossed with the LDL-receptor knockout mouse line ( Ldlr -/- ) and the progenies were fed a high cholesterol diet for 24 weeks. Results showed that R952Q mice were similar to apoER2 knockout mice in displaying increased lesion necrosis compared to Ldlr -/- mice with functional apoER2. However, R952Q mutant mice differed from apoER2 knockout mice, exhibiting increased lipid accumulation in the lesion area. Plasma cholesterol levels were also elevated in the R952Q mutant mice compared to their apoER2 knockout counterparts. Interestingly, unlike the apoER2 deficient VSMCs, the R952Q mutation did not affect VSMC proliferation and showed no signs of accelerated senescence. Thus, the accelerated atherosclerosis observed in the R952Q mutant mice is not due to smooth muscle cell defects, and possibly due to increased plasma cholesterol levels. Additional experiments performed via bone marrow transplant of control and R952Q cells into Ldlr -/- mice showed that the R952Q bone marrow recipients recapitulated the elevated plasma cholesterol levels phenotype of the R952Q mice, suggesting a novel mechanism by which the R952Q mutation in macrophages may accelerate atherosclerosis. Conclusion: The R952Q mutation in apoER2 accelerates atherosclerosis via a gain-of-function mechanism in macrophages with a unique phenotype that exacerbates hypercholesterolemia.

Stanozolol induces ventricular dysfunction by decreasing phospholamban phosphorylation in heart tissue of LDLr-/- mice

Stanozolol is a steroid that causes lipid deposition in LDLr-/- mice, although the mechanism by which this dyslipidemia results in cardiac dysfunction is little understood. The aim of this study was to evaluate the effect of stanozolol on cardiac contractility and the participation of myocardial phospholamban (pPLB) phosphorylation in an atherosclerosis mouse model. LDL receptor knockout mice (LDLr-/-) were fed a standard chow diet and received weekly subcutaneous injections of either saline (control, C group) or 20 mg/kg stanozolol (S group). After 8 weeks, hemodynamic parameters were assessed in the left ventricle. The heart was collected, weighted for hypertrophy evaluation, and kept in formalin buffer for morphometric analysis (H&amp;E) and collagen quantification (Picrossirius). Protein expression of PLB and its phosphorylated form (p-PLB) in the left ventricle was determined by western blot. We observed that stanozolol treatment favored cardiac hypertrophy and collagen deposition in heart tissue. Also, stanozolol induced left ventricle dysfunction, increasing PBL expression and decreasing the p-PLB/PLB ratio. Altogether, our data showed that stanozolol causes cardiac remodeling and ventricular dysfunction by decreasing PLB phosphorylation in the left ventricle of LDLr-/- mice.

In Vivo Pravastatin Treatment Reverses Hypercholesterolemia Induced Mitochondria-Associated Membranes Contact Sites, Foam Cell Formation, and Phagocytosis in Macrophages.

Statins are successful drugs used to treat hypercholesterolemia, a primary cause of atherosclerosis. In this work, we investigated how hypercholesterolemia and pravastatin treatment impact macrophage and mitochondria functions, the key cell involved in atherogenesis. By comparing bone marrow-derived macrophages (BMDM) of wild-type (WT) and LDL receptor knockout (LDLr−/−) mice, we observed hypercholesterolemia increased the number of contact sites at mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), enhanced mitochondrial hydrogen peroxide release, altered the gene expression of inflammatory markers, and increased oxidized LDL (ox-LDL) uptake and phagocytic activity. Three months of in vivo pravastatin treatment of LDLr−/− mice reversed the number of contact sites at the MAM, ox-LDL uptake, and phagocytosis in LDLr−/− BMDM. Additionally, pravastatin increased BMDM mitochondrial network branching. In peritoneal macrophages (PMs), hypercholesterolemia did not change MAM stability, but stimulated hydrogen peroxide production and modulated gene expression of pro- and anti-inflammatory markers. It also increased mitochondrial branching degree and had no effects on ox-LDL uptake and phagocytosis in PM. Pravastatin treatment increased superoxide anion production and changed inflammation-related gene expression in LDLr−/− PM. In addition, pravastatin increased markedly the expression of the mitochondrial dynamics-related genes Mfn2 and Fis1 in both macrophages. In summary, our results show that hypercholesterolemia and pravastatin treatment affect macrophage mitochondria network structure as well as their interaction with the endoplasmic reticulum (ER). These effects impact on macrophage conversion rates to foam cell and macrophage phagocytic capacity. These findings associate MAM stability changes with known mechanisms involved in atherosclerosis progression and resolution.

PRMT3 inhibitor SGC707 reduces triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice

Protein arginine methyltransferase 3 (PRMT3) is a co-activator of liver X receptor capable of selectively modulating hepatic triglyceride synthesis. Here we investigated whether pharmacological PRMT3 inhibition can diminish the hepatic steatosis extent and lower plasma lipid levels and atherosclerosis susceptibility. Hereto, male hyperlipidemic low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet and injected 3 times per week intraperitoneally with PRMT3 inhibitor SGC707 or solvent control. Three weeks into the study, SGC707-treated mice developed severe pruritus and scratching-associated skin lesions, leading to early study termination. SGC707-treated mice exhibited 50% lower liver triglyceride stores as well as 32% lower plasma triglyceride levels. Atherosclerotic lesions were virtually absent in all experimental mice. Plasma metabolite analysis revealed that levels of taurine-conjugated bile acids were ~ threefold increased (P < 0.001) in response to SGC707 treatment, which was paralleled by systemically higher bile acid receptor TGR5 signalling. In conclusion, we have shown that SGC707 treatment reduces hepatic steatosis and plasma triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice. These findings suggest that pharmacological PRMT3 inhibition can serve as therapeutic approach to treat non-alcoholic fatty liver disease and dyslipidemia/atherosclerosis, when unwanted effects on cholesterol and bile acid metabolism can be effectively tackled.

Nobiletin Prevents High-Fat Diet-Induced Dysregulation of Intestinal Lipid Metabolism and Attenuates Postprandial Lipemia.

Nobiletin is a dietary flavonoid that improves insulin resistance and atherosclerosis in mice with metabolic dysfunction. Dysregulation of intestinal lipoprotein metabolism contributes to atherogenesis. The objective of the study was to determine if nobiletin targets the intestine to improve metabolic dysregulation in both male and female mice. Approach and Results: Triglyceride-rich lipoprotein (TRL) secretion, intracellular triglyceride kinetics, and intestinal morphology were determined in male and female LDL (low-density lipoprotein) receptor knockout (Ldlr-/-), and male wild-type mice fed a standard laboratory diet or high-fat, high-cholesterol (HFHC) diet ± nobiletin using an olive oil gavage, radiotracers, and electron microscopy. Nobiletin attenuated postprandial TRL levels in plasma and enhanced TRL clearance. Nobiletin reduced fasting jejunal triglyceride accumulation through accelerated TRL secretion and lower jejunal fatty acid synthesis with no impact on fatty acid oxidation. Fasting-refeeding experiments revealed that nobiletin led to higher levels of phosphorylated AKT (protein kinase B) and FoxO1 (forkhead box O1) and normal Srebf1c expression indicating increased insulin sensitivity. Intestinal length and weight were diminished by HFHC feeding and restored by nobiletin. Both fasting and postprandial plasma GLP-1 (glucagon-like peptide-1; and likely GLP-2) were elevated in response to nobiletin. Treatment with a GLP-2 receptor antagonist, GLP-2(3-33), reduced villus length in HFHC-fed mice but did not impact TRL secretion in any diet group. In contrast to males, nobiletin did not improve postprandial lipid parameters in female mice. Nobiletin opposed the effects of the HFHC diet by normalizing intestinal de novo lipogenesis through improved insulin sensitivity. Nobiletin prevents postprandial lipemia because the enhanced TRL clearance more than compensates for increased TRL secretion.

Therapeutic Effects of Fenofibrate Nano-Emulsion Eye Drops on Retinal Vascular Leakage and Neovascularization.

Simple SummaryAge-related macular degeneration (AMD) and diabetic retinopathy (DR) are the leading causes of blindness and lack non-invasive drug treatments. Macular edema and neovascularization are the key pathogenic features responsible for vision loss in AMD and DR. Clinical studies showed that fenofibrate, a PPARα agonist, has robust therapeutic effects on macular edema and retinal neovascularization after oral administration. However, the efficacy of oral fenofibrate remains to be improved, due to limited amounts of the drug delivered to the retina after systemic administration. To increase the drug availability in the retina, this study developed a novel nano-emulsion fenofibrate eye drop. Topical administration of the fenofibrate eye drop delivered significantly higher drug concentrations to the retina/vitreous, relative to the systemic administration. The fenofibrate eye drop alleviated retinal inflammation and vascular leakage in both DR and AMD models. Further, the fenofibrate eye drop also alleviated laser-induced choroidal neovascularization. These findings suggest that topical administration of the nano-emulsion-based eye drop of a PPARα agonist has potential to become a non-invasive therapeutic strategy for long-term treatment of DR and AMD.Macular edema caused by retinal vascular leakage and ocular neovascularization are the leading causes of severe vision loss in diabetic retinopathy (DR) and age-related macular degeneration (AMD) patients. Oral administration of fenofibrate, a PPARα agonist, has shown therapeutic effects on macular edema and retinal neovascularization in diabetic patients. To improve the drug delivery to the retina and its efficacy, we have developed a nano-emulsion-based fenofibrate eye drop formulation that delivered significantly higher amounts of the drug to the retina compared to the systemic administration, as measured by liquid chromatography–mass spectrometer (LC-MS). The fenofibrate eye drop decreased leukocytes adherent to retinal vasculature and attenuated overexpression of multiple inflammatory factors in the retina of very low-density lipoprotein receptor knockout (Vldlr−/−) mice, a model manifesting AMD phenotypes, and streptozotocin-induced diabetic rats. The fenofibrate eye drop also reduced retinal vascular leakage in these models. The laser-induced choroidal neovascularization was also alleviated by the fenofibrate eye drop. There were no detectable ocular toxicities associated with the fenofibrate eye drop treatment. These findings suggest that fenofibrate can be delivered efficiently to the retina through topical administration of the nano-emulsion eye drop, which has therapeutic potential for macular edema and neovascularization.

The Cordyceps militaris-Derived Polysaccharide CM1 Alleviates Atherosclerosis in LDLR(-/-) Mice by Improving Hyperlipidemia.

Atherosclerotic cardiovascular disease has a high mortality worldwide. Our lab previously purified a polysaccharide designated as CM1 with (1→4)-β-D-Glcp and (1→2)-α-D-Manp glycosyls as the backbone. In this study, we investigated the anti-atherosclerosis effect of CM1 and the underlying mechanisms of action in a low-density lipoprotein receptor knockout (LDLR(-/-) mouse model. It was found that CM1 significantly decreased the formation of atherosclerotic plaques. Mechanistically, CM1 enhanced plasma level of apolipoprotein A-I and decreased the plasma levels of triglyceride, apolipoprotein B, and total cholesterol. In the absence of LDLR, CM1 elevated the expression of very low-density lipoprotein receptor for liver uptake of plasma apolipoprotein B-containing particles and reduced hepatic triglyceride synthesis by inhibiting sterol regulatory element binding protein 1c. CM1 improved lipids excretion by increasing the liver X receptor α/ATP-binding cassette G5 pathway in small intestine. CM1 reduced lipogenesis and lipolysis by inhibiting peroxisome proliferator-activated receptor γ and adipose triglyceride lipase in epididymal fat. Furthermore, CM1 improved lipid profile in C57BL/6J mice. Collectively, CM1 can modulate lipid metabolism by multiple pathways, contributing to reduced plasma lipid level and formation of atherosclerotic plaques in LDLR(−/−) mice. This molecule could be explored as a potential compound for prevention and treatment of hyperlipidemia and atherosclerosis.