High Cholesterol Diet Research Articles

Cardiorenal protective effects of extracts of bitter leaf (Vernonia amygdalina) in animal model of metabolic syndrome

Every year, metabolic syndrome and cardiorenal diseases cause many deaths worldwide. African bitter leaf (Vernonia amygdalina) is known for its numerous therapeutic effects. Potentially, it can lower plasma lipid and glucose levels, which, in turn, may improve the condition of patients with the abovementioned diseases. This research featured the antihyperlipidemic and antihyperglycemic effects of methanol extract of V. amygdalina in an animal model of metabolic syndrome.
 Twenty albino rats were divided into four groups. Groups A to C were orally administered with ghee (3 mL/kg) + high-cholesterol diet (500 mg/kg) + high-sugar diet (10 mL/kg) to induce metabolic syndrome. Group A served as negative control and received no treatment with bitter leaf methanol extract. Groups B and C received 200 and 400 mg/kg of V. amygdalina methanol extract, respectively. Group D received no administration. The cardiorenal injuries and alterations in blood lipids and sugar levels wereassessed via various biochemical analyses.
 The combination of ghee + high-cholesterol diet + high-sugar diet triggered a significant elevation of creatine kinase myocardial band, lactate dehydrogenase, aspartate aminotransferase, triglycerides, total cholesterol, low density lipoprotein-cholesterol, glucose, urea, creatinine, and potassium levels. The histopathological results agreed with the biochemical findings. However, the treatment with 200 and 400 mg/kg of V. amygdalina methanol extract was able to inhibit the adverse alterations causing a dosedependent significant antihyperlipidemic and antihyperglycemic effect (p < 0.05).
 Bitter leaf (V. amygdalina) demonstrated cardiorenal protective effects and may be used to manage metabolic syndrome.

Therapeutic effect of yinchenhao decoction on cholelithiasis via mucin in the gallbladder and intestine

Cholelithiasis is a common and frequently occurring disease worldwide that belongs to the category of jaundice in traditional Chinese medicine. Yinchenhao decoction (YD) consists of Artemisia capillaris Thunb., Gardenia jasminoides J.Ellis, and Rheum palmatum L., and is traditionally used to treat jaundice, which has a significant therapeutic effect on cholelithiasis. Our study aimed to investigate the pathological mechanism of cholelithiasis and the therapeutic mechanism of YD via mucin in the gallbladder and intestine. YD was prepared and analyzed using HPLC. The supersaturation stability experiment was designed by the solvent-shift method. The cell transport experiment was conducted by coculture monolayers. The animal experiment was performed using a cholelithiasis model with a high-cholesterol diet. The related indicators were detected by automatic biochemical analyzer, PCR, western blot, or ELISA. Statistics were analyzed using χ2-tests and t-tests. As the results, in cholelithiasis, MUC5AC highly expressed in the gallbladder shortened cholesterol supersaturation and promoted cholesterol crystallization via the inflammatory cytokine signaling pathway; MUC2 highly expressed in the small intestine prolonged cholesterol supersaturation and promoted cholesterol absorption via the inflammatory cytokine signaling pathway. YD inhibited mucin expression in the gallbladder and intestine in a concentration-dependent manner for cholelithiasis treatment by inhibiting the inflammatory cytokine signaling pathway, which was attributed to the active components, including chlorogenic acid, geniposide, and rhein.

Role of S100A4 in the crosstalk between smooth muscle and inflammatory cells in atherosclerosis

Abstract Background During atherosclerotic plaque formation, smooth muscle cells (SMCs) accumulate in the intima and undergo a transition from a contractile to a synthetic phenotype. We previously showed that S100A4 is a marker of the synthetic SMCs in vitro and intimal SMCs in pig, human and mouse. In vitro, S100A4-treated SMCs acquire a pro-inflammatory phenotype. The systemic neutralization of extracellular S100A4 in ApoE-/- mice induces atherosclerotic plaque stabilization. Purpose To characterize the role of S100A4-expressing SMCs in atherosclerosis. Methods and results We established a coculture model between S100A4-stimulated human aortic SMCs and human blood-derived monocytes. Flow cytometry analysis of monocytes exposed to S100A4-treated SMCs for 3 days showed an upregulation of markers typical of inflammatory M1 (HLA-DR) and anti-inflammatory M2 (CD206) macrophages (fig. 1). Macrophages also upregulated mRNA expression of NPC Intracellular cholesterol transporter 1 and 2 by qPCR, suggesting modifications in their cholesterol metabolism. In vivo, in an SMC-lineage-tracing ApoE-/-mouse model, we identified S100A4-expressing SMCs at the onset of atherosclerosis in aortic media of 11-week-old mice fed a high cholesterol diet (HCD) for 3 weeks. Medial SMCs were characterized by means of single-cell RNA sequencing. Interestingly, 10 out of 11 identified clusters expressed S100A4, demonstrating broad and early involvement of S100A4 in shaping medial SMC identity. We assumed that blood-derived monocytes might be a driving force of early S100A4 activation in SMCs. To mitigate the impact of monocytes, 8-week-old ApoE-/- mice were fed an HCD for 9 weeks and injected with either clodronate-encapsulated or empty liposomes during the first 3 weeks. No differences in S100A4 expression in the media were observed by qPCR. Unexpectedly, ApoE-/- mice treated with clodronate demonstrated larger atherosclerotic lesions compared to control animals as measured by OilRedO staining (fig. 2). Studies are in progress to define the plaque composition. Conclusion Our findings shed light on the role of S100A4 in the crosstalk between SMCs and inflammatory cells in atherosclerosis and the fine-tuning of the phenotype of medial SMCs prior to their accumulation in the intima. We suggest that temporary monocyte depletion at the onset of the disease alters the mechanisms underlying plaque formation, worsening the outcome of the disease, while not affecting S100A4 expression in medial SMCs.

A single-cell atlas of immune checkpoint molecules in atherosclerosis in ApoE-/- mice

Abstract Introduction Immune checkpoint (IC) targeting is an attractive way to combat atherosclerosis, since ICs regulate the immune response by activating or inhibiting inflammatory pathways. We have shown that ICs exert cell-divergent functions and that cell-specific IC-based treatments can limit immune-related side effects. To design tailored IC-based immunotherapies for atherosclerosis, a detailed single-cell atlas of IC expression during atherogenesis is required. Purpose To obtain cell-type- and atherosclerosis-stage-specific expression profiles of ICs and assess the transcriptome of IC+ cells during atherosclerosis. Methods ApoE-/- mice were fed normal chow diet (NCD) for 10, 20 or 30 weeks, or high-cholesterol diet (HCD) for 6, 10, 14 (study 1) or 20 weeks (study 2). Using mass-cytometry by time of flight (CyTOF), we assessed the cell-type-specific aortic expression of ICs during atheroprogression (study 1). Using cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq), we assessed the transcriptome of aortic IC+ and IC- cells (study 2). Results Using CyTOF, we identified 21 immune cell clusters, including T and B cells, Ly6C- and Ly6C+ monocytes and macrophage (mφ) subsets. On T cell subsets, IC expression remained relatively constant in early plaques independent of the type of diet, apart from an increase in CD40-CD40L and decrease in CD28 and PD-L1 in HCD-fed mice. In advanced plaques from NCD-fed mice, most T cell subsets showed increased PD-L1, GITRL and CD27, while in HCD-fed mice, CD40L was decreased and PD-L1, PD-L2, CD27, CD86-CD28 and GITR-GITRL were induced on these T cell subsets. On myeloid cells, IC expression changed during early atherogenesis. Specifically, in NCD-fed mice, CD11c+MHCII+ mφ upregulated CD40 and PD-L1 and downregulated CD27 and PD-L2, while dendritic cells increased GITR and decreased PD-L2. Additional ICs, such as GITRL, CD40L and CD86-CD28, were induced in advanced plaques from NCD-fed mice. In HCD-fed mice, numerous ICs, i.e. CD40-CD40L, GITR-GITRL, CD86-CD28 and PD-L2, were increased in myeloid subsets in both early and advanced plaques. Using CITE-Seq, we further studied ICs that were strongly regulated during atherogenesis, e.g. GITR. Gene Ontology enrichment analysis of GITR+ and GITR- cells within the myeloid and T cell subsets identified the regulation of various pathways: When compared to their GITR- counterparts, both CD25- and CD25+ CD4+GITR+ T cell subsets showed upregulated pathways of death receptor activity, while CD25+ cells also showed induction of pathways involved in transmembrane lipid transporter activity. In mφ, GITR+ mφ showed upregulated pathways of cell adhesion and cytokine activity compared to GITR- mφ. Conclusion We have identified the expression pattern of numerous ICs on aortic immune cell subsets at various atherosclerosis stages. These data will pave the way towards the development of selective IC-based therapies to combat atherosclerosis.

High dose NAD+ boosting as pan-sirtuin activation increases atherosclerotic plaques and systemic inflammation in Apoe knockout mice

Abstract Background NAD+ (nicotinamide adenine diphosphate) is a cosubstrate of sirtuins (SIRT) that are activated upon caloric restriction. The nuclear SIRT1 and SIRT6 exert protective effects on atherothrombosis through immunometabolic pathways. By supplementing its precursors, an increase in the NAD+ pool has been reported to extend life span in mice. However, supplementing NAD+ precursors may also stimulate inflammation depending on the context. Nicotinamide riboside (NR) is a precursor of NAD+, whose supplementation has been used to combat metabolic syndromes and liver steatosis. Notably, the effects of pan-sirtuin activation using NR on atherosclerosis remain unknown. Purpose We hypothesized that NR supplementation confers atheroprotection by activating the NAD+ sirtuin axis. We aimed to test this hypothesis by investigating the effects of NR supplementation on plaque formation using an atherosclerosis-prone mouse model. Methods 8-week-old male apolipoprotein E (ApoE) knockout (KO) mice were fed for 12 weeks a high-cholesterol diet (HCD, 1.25 wt%) supplemented with three NR doses: 0, 1.2, and 2.4 g/kg diet. The groups were designated as NR-, NR+, and NR++, respectively (n=14/group). After overnight fasting, aortae, liver, and blood were collected. Cytokines were analyzed using a multiplex ELISA. Triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) levels were determined by colorimetry and the Friedewald equation. Cross sections of the aortic root were stained with Oil-Red O. Results High-dose (HD) NR supplementation increased plaque thickness in NR++ ApoE KO mice, compared with NR+ mice (Figure 1A and B). In parallel, plasma expression levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 as well as TG and LDL-C levels were increased in NR++ mice (Figure 1C-F) suggesting that NR supplementation induced systemic inflammation and disturbed lipid metabolism. Analyses of liver lysates showed that expression levels of SIRT1 were decreased and CD38 increased in NR++ mice. Full length PARP1 remained unchanged, while both cleaved PARP1 and total PARylation in liver decreased. NAD+ concentrations in liver and plasma remained unchanged. These data suggest that an increase in hepatic CD38 might consume more NAD+ in the atherosclerotic environment and could be the culprits for worsening atherosclerotic progression and systemic inflammation (Figure 2). Conclusions HD NR supplementation increased plaque thickness, as well as IL-6 and LDL-C plasma levels, moderate NR dose had neutral effects. Elevated hepatic expression levels of CD38 and decreased SIRT1 may at least in part explain the atherosclerotic phenotype. Thus, HD NR supplementation in mice does not decrease but worsen atherosclerosis and induce systemic inflammation. A cautious approach should be considered when applying NAD+ boosting in patients with atherosclerotic disease.

MicroRNA-206-3p suppresses hepatic lipogenesis and cholesterol synthesis while driving cholesterol efflux.

Hepatosteatosis, hypertriglyceridemia, and hypertriglyceridemia are interconnected metabolic disorders. This study is designed to characterize how microRNA-206-3p (miR-206) simultaneously prevents de novo lipogenesis (DNL), cholesterol synthesis, and VLDL production in hepatocytes while promoting cholesterol efflux in macrophages. MiR-206 levels were reduced in hepatocytes and macrophages of mice subjected to a high-fat, high-cholesterol diet (HFHC). A negative feedback between LXRα (liver X receptor) and miR-206 is formed to maintain high LXRα and low miR-206 in hepatocytes. Systemic administration of miR-206 alleviated hepatosteatosis, hypertriglyceridemia and hypercholesterolemia in mice. A significant reduction in LDL-cholesterol and VLDL-cholesterol but unaltered HDL-cholesterol was observed in miR-206-treated mice. Mirroring these findings, miR-206 reprogrammed the transcriptome of hepatocytes towards inhibition of DNL, cholesterol synthesis, and assembly and secretion of VLDL. In macrophages, miR-206 activated expression of genes regulating cholesterol efflux. Hepatocyte-specific expression of miR-206 reduced hepatic and circulating triglycerides and cholesterol as well as VLDL production, while transplantation of macrophages bearing miR-206 facilitated cholesterol efflux. Mechanistically, miR-206 directly targeted Lxrα and Hmgcr in hepatocytes but facilitated expression of Lxrα in macrophages by targeting macrophage-specific TRPS1 (tricho-rhino-phalangeal syndrome 1), a transcription repressor of Lxrα. By targeting Hmgcr and Lxrα, miR-206 inhibited DNL, VLDL production and cholesterol synthesis in hepatocytes, whereas it drove cholesterol efflux by activating the TRPS1-LXRα axis. MiR-206, through differentially modulating LXRα signaling in hepatocytes and macrophages, inhibits DNL, promotes cholesterol efflux, and concurrently hinders cholesterol synthesis and VLDL production. MiR-206 simulates the functions of lipid-lowering medications, statins and LXRα agonists.

Immune system humanization in dyslipidemic Rag2-KO/IL2rg-KO/CD47-KO/LDL-R KO mice controls atherosclerosis

Abstract Background and aim Given the key role of the immune response during atherosclerosis and the therapeutic interest of biologics targeting human immune cells, the need of experimental models to translate molecular mechanisms and to test therapeutic approaches for atherosclerosis is continuously increasing. Here we describe the characteristics of an innovative immunodeficient mouse humanized with hCD34+ cells on an atheroprone background. Methods LDLR-KO mice were crossed with the immunodeficient C57BL/6J strain Rag2-KO/IL2rg-KO/CD47-KO (TKO, IMSR_JAX:025730) to generate an immunocompromised dyslipidemic mouse recipient TKO-LDLR KO of human hematopoietic stem cells (hCD34+). Results TKO-LDLR KO were first characterized for their immune and metabolic profile. TKO mice are deficient in mature lymphocytes and NK cells and this profile was conserved in TKO-LDLR KO mice. Under high cholesterol diet for 8 weeks, both male and females TKO-LDLR KO present monocytosis with increased levels of Ly6Chi monocytes compared to TKO-LDLR KO at standard diet, develop marked dyslipidemia (total cholesterol 870.9 and 890.1 mg/dL male and females respectively), steatosis and atherosclerosis. This profile confirms the suitability of TKO-LDLR KO mice for atherosclerosis studies. Next we tested the impact of immune system humanization on atherosclerosis. TKO-LDLR KO pups received a low-dose irradiation (150-200 cGy) and thereafter 1,5-2 x 10^5 hCD34+ were injected with in the liver. Engraftment of human leukocytes (hCD45+) was evaluated after two months by flow cytometry analysis from tail blood. This approach allows to reconstitute between 10-30% of hCD45+, mainly B and T cells. The humanization with hCD34+ cells affected atherosclerosis development as compared to non-humanized TKO-LDLR KO mice. Conclusion We have generated and characterized for the first time a humanized dyslipidemic TKO-LDLR KO mice. This mouse model presents human B and T cells and represent and important toll to investigate the impact of biologics targeted toward human targets in the context of atherosclerosis.

The role of EZH2 and H3K27me3 epigenetic signature in modulating T-cell polarization in atherosclerosis

Abstract Background Atherosclerosis is a chronic inflammatory disease, characterized by the formation of luminal plaques in arteries. Atheroprogression is accompanied by strong immune activation, during which T cells play a pivotal role. Genes driving T-cell activation are regulated by the polycomb repressive complex 2(PRC2), whose major component is the Enhancer of Zeste Homolog 2(EZH2), responsible for the methylation of Histone 3 Lysine 27(H3K27me3) epigenetic mark. Purpose We hypothesize that EZH2 mediates T-cell activation and modulates T-helper (Th) cell polarization, thus atherogenesis. Methods To study the effect of EZH2 in atherosclerosis, we backcrossed mice with Ezh2 flanked by loxP-sites sensitive to Cre-mediated inactivation in T cells to apolipoprotein E (Apoe-/-)mice and fed a high cholesterol diet to have hyperlipidemic Ezh2fl/flCd4CreApoe-/-mice and controls. Atherosclerotic plaque progression and immune cell phenotyping were assessed by histology, flow cytometry (FC), gene and protein expression analysis and single cell (sc)RNA-sequencing (Seq) as well as by functional in vitro assays. Results T cell-specific EZH2 deficiency resulted in a significant reduction in H3K27me3 in T-cells. Female Ezh2fl/flCd4Cre mice developed 34% less atherosclerotic plaques in the aortic root compared to wildtype (WT) littermates. FC analysis of spleen and lymph nodes of Ezh2fl/flCd4Cre mice revealed a shift from naive to effector CD4+ T cells, and the majority were Th2 cells (p<0.0001). Accordingly, plasma levels of lL4 and aortic IL4 transcripts were increased in Ezh2fl/flCd4Cre mice(p=0.05 and p=0.0079, respectively). Functional analysis revealed that splenic CD4+ T cells from Ezh2fl/flCd4Cre mice showed decreased migratory capacity towards the chemokines CXCL10, CCL17, CCL22, CCL19. To unravel mechanisms driving the type 2 immune response, bulk RNA-Seq of CD4+ T cells and scRNA-Seq of splenic CD3+ T cells from Ezh2fl/flCd4Cre and WT mice revealed that CD4+ T cell EZH2 deficiency caused upregulation of promyelocytic leukemia zinc finger (Plzf), a transcription factor of NKT cells of the NKT2 subtype, characterized by high IL4 production. Via FC we confirmed the increase in splenic NKT cells in Ezh2fl/flCd4Cre mice. Likewise, Plzf aortic mRNA levels were also significantly increased(p=0.002). To exploit the translational potential of T cell-specific EZH2 targeting, we treated Jurkat T cells with EZH2 inhibitors, the FDA-approved drug Tazemetostat and GSK126. Corroborating our in vivo data, EZH2 inhibitor-treated cells showed decreased H3K27me3 and 70% reduced migration towards the chemokine CXCL12. Conclusions Our study demonstrates that CD4+ T-cell specific EZH2 deficiency impairs T cell-migration and skews the immune response towards a type 2 response, resulting in protection against atherosclerosis. We aim at investigating whether the epigenetic mark H3K27me3 in T cells may be a promising therapeutic target for future cardiovascular immunotherapies.

Macrophage expressed CD36 promotes plaque vulnerability in atherosclerosis

Abstract Background Atherosclerosis is characterised by a chronic lipid-driven inflammation that accounts for the majority of deaths worldwide. CD36 is an immunoregulatory scavenger receptor at the interface of lipid metabolism and inflammation that is critically involved in atherogenesis. However, due to the lack of cell-specific in vivo models the exact role of CD36 on macrophages in atherosclerosis remains unknown to date. Purpose Here we aimed to evaluate the role of macrophage expressed CD36 in atherogenesis. Approach & Results: To screen for a potential role of macrophage expressed CD36 in atherosclerosis we assessed CD36 gene expression across different leukocyte populations in single-cell-RNA Sequencing (scRNAseq) data from atherosclerotic LDLR-/- mice. Here, we identified macrophages as the main CD36 expressing cell type. In line, CD36 protein expression in aortic cell suspensions from LDLR-/- mice was significantly higher in macrophages compared to B and T cells as assessed by flow cytometry. To assess the role of macrophage expressed CD36 in atherogenesis, we generated macrophage specific CD36 deficient mice on an atherosclerotic prone LDLR deficient (LDLR-/-) background. CD36fl/fl-CXC3R1Cre LDLR-/- mice (termed iMΦ-CD36) and CD36fl/fl-CXC3R1Wt LDLR-/- controls (termed CD36fl/fl) were injected with tamoxifen for 5 consecutive days and placed on a high cholesterol diet for 16 weeks to induce atherogenesis. At harvest the aortic root was obtained from whole heart, embedded in OCT, cut and stained with Oil Red O staining for histological analyses of atherosclerotic lesions. Macrophages were differentiated from isolated bone marrow cells for in vitro assessment of ROS production and oxLDL uptake. Notably, Macrophage-specific CD36 deficiency did not affect atherosclerotic lesion size but substantially reduced the necrotic core area indicating ameliorated plaque vulnerability. In vitro, macrophage CD36 deficiency ameliorated ROS production and facilitated oxLDL uptake. Further, macrophage-specific CD36 deficiency reduced circulating MCP-1 levels. To interrogate a potential role of CD36 in human atherosclerosis, human atherosclerotic plaques were studies by scRNAseq. Similar to our observations in mice, cellular CD36 expression was highest in human macrophages/monocytes. Further pathway analysis revealed that CD36-expressing macrophages exhibited a significantly more pro-inflammatory gene signature than CD36-negative macrophages. Down this line, CD36 expression was significantly higher in unstable carotid plaques compared to stable lesions indicating that CD36 expression correlates with plaque vulnerability in humans. Conclusion All in all, our data indicates a pro-inflammatory role of macrophage expressed CD36 in murine and human atherosclerosis. These findings suggest that targeting CD36 on macrophages may represent a potential target for anti-atherosclerotic therapy.

The role of endomembrane homeostasis in vascular calcification

Abstract Background Cardiovascular diseases are a leading cause of mortality, with cardiovascular calcification as a prominent predictor and contributor. Microcalcifications in thin fibrous caps of atherosclerotic plaques create biomechanical instability, leading to plaque vulnerability. Recent work indicated that cellular-derived extracellular vesicles (EVs) are pivotal for microcalcification nucleation. EVs can originate from the endolysosomal system, and FYVE-Type Zinc Finger Containing Phosphoinositide Kinase (PIKfyve), a lipid kinase, plays a key role in the endolysosomal maturation. Purpose We hypothesize that alterations in endomembrane homeostasis will modulate EV cargo and, thereby, vascular calcification. Methods and Results In calcified arteries and vascular smooth muscle cells (SMCs), we observed increased PIKfyve expression. Phosphatidylinositol 3-phosphate (PI3P) - the substrate of PIKfyve - was decreased in cellular membranes of calcifying SMCs (-40%) and recovered by Apilimod, a small molecule PIKfyve inhibitor. Apilimod prevented matrix mineralization and collagen network formation in calcifying SMCs (Fig. 1A) accompanied by reduced pro-collagen 1A1 secretion (-90%). Apilimod inhibited tissue non-specific alkaline phosphatase (TNAP) – an early marker of SMC calcification on mRNA, protein and activity level. PIKfyve silencing using siRNA showed similar results. Furthermore, Apilimod caused a dose-dependent increase of EV release assessed by nanoparticle tracking analysis (Fig. 1B). Analysis of the EV cargo demonstrated that Apilimod reduces TNAP protein abundance and activity (-60%) - a characteristic cargo that may determine EV calcification potential. Apilimod-induced EVs exhibited lower mineral content and reduced aggregation potential assessed by osteosense-based flow cytometry and turbidity assays. Next, we performed transcriptome and kinome analyses to discover the underlying mechanism of PIKfye-mediated SMC calcification. These data revealed a link to adipocyte-like differentiation and SMC phenotype specifying pathways. Validation supported increased expression of adipogenic transcription factors (CEBPA, PPARG),genes of the cholesterol and fatty acid metabolism pathways (FABP3, CD36) and enhanced fatty acid (C12) uptake (Fig. 1A) by Apilimod; while SMC-specific markers (ACTA2, SM22) and key SMC transcription factors (GATA6, MYOCD) were downregulated. Finally, in Ldlr-deficient mice fed a high-fat, high-cholesterol diet for 15 weeks, Apilimod application for 5 weeks increased PI3P levels (1.6-fold) and decreased vascular calcification (Fig. 1C), but did not alter plaque size or collagen content in atherosclerotic plaques. Conclusion Disrupting endolysosomal maturation with Apilimod promotes the release of EVs with reduced calcification potential and induces a phenotypic adaption towards adipocyte-like SMCs, causing reduced SMC calcification. Anti-arteriosclerotic effects of Apilimod in vivo remain to be further investigated.Figure

Sirtuin-1 directly binds and deacetylates hepatic PCSK9 to inhibit low-density lipoprotein receptor degradation

Abstract Background Low-density lipoprotein cholesterol (LDL-C) is causally involved in atherosclerotic cardiovascular disease (ASCVD). We have previously shown that pharmacological activation of Sirtuin-1 (SIRT1), a NAD+-dependent deacetylase, reduces plasma LDL-C levels by decreasing PCSK9 and increasing hepatic LDLR expression, thus exerts atheroprotective effects in mice; however, the mechanism of SIRT1 action remains elusive. Purpose We found diminished levels of circulating SIRT1 in atherosclerotic mice compared to wild-type healthy controls. We hypothesized that this decrease in SIRT1 levels may promote dysregulated lipid metabolism and ASCVD in humans. Herein, we aimed to restore the reduced systemic Sirtuin-1 plasma levels in ASCVD by injecting recombinant murine Sirtuin-1 (rmSIRT1) in atherosclerotic mice. Methods Twelve-week-old apolipoprotein E-deficient (ApoE-/-) male mice fed a high-cholesterol diet (1.25% w/w) were randomized to receive rmSIRT1 (n=6; 0.3mg/kg BW i.p.) or vehicle treatment (n=6) every third day over 4-weeks. Mice were euthanized and organs were harvested to study lipid metabolism and ASCVD progression. Human hepatocytes (Huh7) cells were used to perform radioactive assays to study cellular uptake of radioiodinated LDL, surface plasmon resonance to study binding and site-directed mutagenesis to dissect the underlying molecular mechanism exerted by circulating SIRT1. Results Boosting circulating SIRT1 levels increased hepatic LDLR expression, reduced plasma LDL-C levels and progression of plaques in ApoE-/- mice (Figure 1). rmSIRT1 treatment did not change hepatic expression of total PCSK9 but increased its deacetylated status. Mechanistically, rmSIRT1 directly bound to hepatic PCSK9 and deacetylated PCSK9 at 3 sites: Lys243, Lys421 and Lys506 as shown by Surface Plasmon Resonance and mass spectrometry, respectively. In vitro mutagenesis to triple deacetylation mimetic (3KR) in hepatocytes (Huh7 cells) reduced SIRT1-induced PCSK9 activity, as evidenced by increased cellular binding and association of 125I-LDL through LDLR in Huh7cells. Finally, plasma levels of SIRT1 and PCSK9 were assessed at baseline in patients with acute coronary syndromes (ACS) where plasma SIRT1 showed an inverse correlation with PCSK9 and conferred a reduced risk of major adverse cardiovascular events. Conclusion SIRT1 directly binds PCSK9 and decreases its activity by deacetylation, thereby enhancing LDL-C clearance by hepatic LDLR upregulation. Increased circulating SIRT1 exerts atheroprotective effects and is associated with improved prognosis in patients with ACS.Figure 1

Colchicine prevents accelerated atherosclerosis development in TET2-mutant clonal hematopoiesis

Abstract Background Somatic mutations in the TET2 gene that lead to clonal hematopoiesis are associated with accelerated atherosclerosis development in mice and a higher risk of atherosclerotic cardiovascular disease events in humans. Mechanistically, these observations have been linked to exacerbated vascular inflammation. Purpose Here, we sought to evaluate whether colchicine, a widely available and inexpensive anti-inflammatory drug, prevents the accelerated atherosclerosis associated with TET2-mutant clonal hematopoiesis. Methods In mice, we mimicked TET2-mutant clonal hematopoiesis using a competitive bone marrow transplantation approach in atherosclerosis prone Ldlr/- mice. We generated hematopoietic chimeras carrying initially 10% Tet2-/- hematopoietic cells, which were fed a high-cholesterol diet and treated with colchicine or placebo. Atherosclerosis development was evaluated histologically. In humans, we analyzed whole exome sequencing data and electronic medical records from 50,291 participants in a hospital-based cohort to examine the potential modifying effect of colchicine prescription on the association between clonal hematopoiesis and myocardial infarction. Results Colchicine prevented the development of accelerated atherosclerosis in the mouse model of TET2-mutant clonal hematopoiesis, without affecting serum cholesterol levels or the expansion of TET2-deficient hematopoietic cells. In humans, consistent with experimental findings, patients who were prescribed colchicine had attenuated associations between TET2-mutant clonal hematopoiesis and myocardial infarction (ORno colchicine [95% CI]: 1.18 [0.93-1.48]; ORcolchicine [95% CI]: 0.78 [0.45-1.35]; Pinteraction=0.03). This interaction was not observed for carriers of other clonal hematopoiesis-related mutations, such as those affecting DNMT3A or ASXL1. Conclusions Our results highlight the potential value of colchicine as a targeted preventive care strategy to mitigate the higher cardiovascular risk of carriers of somatic TET2 mutations in blood cells. These observations set the basis for the development of clinical trials that evaluate the efficacy of precision medicine approaches tailored to the effects of specific somatic mutations linked to clonal hematopoiesis.

Abstract 18666: SR-BI and Tyrosine Kinase Inhibitors: A Novel Pathway for Atherosclerotic Cardiovascular Disease Therapeutic Development

Introduction: Cardiovascular disease (CVD) is the leading cause of death in the U.S., where coronary artery disease (CAD) accounts for 42.1% of all CVD deaths. Current therapeutics focus on lowering LDL-C, as previous attempts to raise HDL-C were not successful in altering CVD outcomes. However, the therapeutic potential of HDL has not been fully explored. Variants in SCARB1, the gene that encodes HDL receptor Scavenger Receptor B1 (SR-BI), are associated with dyslipidemia and atherosclerotic CVD (ASCVD), and we first to identified Mendelian inheritance of SCARB1 variants that cause severe early-onset CAD and dyslipidemia. Limited clinical evidence suggests imatinib reduces total cholesterol in chronic myeloid leukemia (CML). Additionally, TKI treatment in atherosclerotic mice reduced total cholesterol (imatinib) and atherosclerotic lesions (dasatinib). Yet, no data is available on the effects of TKIs on HDL and RCT. Hypothesis: Our findings suggest that HDL function (vs. HDL-C concentration) in reverse cholesterol transport (RCT) may be a promising target for cholesterol-based therapy and studying TKIs in RCT may provide a novel mechanism for therapeutic development. Methods: We performed a high throughput drug screen with 788 FDA-approved compounds, using HepG2 cells to measure endogenous HDL binding. We identified four compounds that significantly increased HDL binding, of which, imatinib was the only one to increase SR-BI. Wildtype C57Bl/6 mice on a high fat, high cholesterol diet, underwent imatinib treatment for 4 weeks and measured biweekly serum lipids. Results: We have found that imatinib and dasatinib significantly enhance HDL binding, where imatinib increased SR-BI, ABCA1, and ABCG1 in vitro. Furthermore, in vivo imatinib treatment decreased plasma total cholesterol, HDL-C and triglyceride levels, and elevated hepatic SR-BI. Knockdown of SR-BI in HepG2 cells ameliorated the imatinib-induced HDL binding increase suggesting an SR-BI-specific mechanism. Mass spectrometry revealed a novel SR-BI methionine oxidation site with imatinib treatment. Conclusions: Our data supports the exploration of TKI-mediated SR-B1 regulation, HDL metabolism, and RCT mechanism to identify new therapeutic targets for dyslipidemia and ASCVD.

Abstract 17384: Unraveling the Impact of VAMP-8 Deficiency on Atherogenesis: A Fresh Perspective on Platelet Involvement

Introduction and Objective: Recognized for their intricate role in atherogenesis, platelets interact with oxidized low-density lipoprotein (oxLDL), immune cells, and endothelial cells and release granule proteins and microparticles, contributing to the onset and advancement of atherosclerosis. Despite the challenges of antiplatelet treatment in primary prevention, exploring alternative platelet targets could provide key insights into their role in atherogenesis. This study pivots on the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins, notably vesicle associated membrane protein 8 (VAMP-8) - the primary vesicle membrane (v-SNARE) and vital facilitator of platelet secretion. Approach and Results: The profound impairment in α-granule, dense granule, and lysosome release due to VAMP-8 deficiency directed our investigation towards its potential role in atherogenesis. Using RNA-seq data from washed platelets of VAMP-8-/- mice versus wildtypes, we first analyzed the molecular signature of VAMP-8 deficiency. Gene ontology analysis revealed that the major differences in enriched genes between the two groups involved triglyceride biosynthetic process and platelet calcium homeostasis. We initiated atherosclerosis studies using ApoE -/- mice crossed with VAMP-8 -/- mice (n=11) alongside littermate controls (n=9), all fed a high cholesterol diet for 15 weeks. Remarkably, the deletion of VAMP-8 led to a substantial reduction in atherosclerotic lesion development when compared to the control group. Notably, this phenotype manifested irrespective of total cholesterol levels, as plasma cholesterol remained consistent across all groups. Conclusion: Our findings reveal that VAMP8 deficiency markedly mitigates atherogenesis, thereby proposing a novel perspective to explore the influence of platelet cargo secretion on the inception of atherosclerosis. We are currently conducting additional studies to uncover the underlying mechanisms responsible for these observations

Abstract 17224: Macrophage Expressed CD36 Promotes Plaque Vulnerability in Atherosclerosis

Introduction: Atherosclerosis is characterised by a chronic lipid-driven inflammation that accounts for the majority of deaths worldwide. CD36 is an immunoregulatory scavenger receptor at the interface of lipid metabolism and inflammation that is critically involved in atherogenesis. However, due to the lack of cell-specific animal models the exact role of CD36 on macrophages in atherosclerosis remains unknown to date. Research Question: Here, we aimed to study the role of macrophage expressed CD36 in atherogenesis. Methods: To screen for a potential role of macrophage expressed CD36 in atherosclerosis we assessed CD36 gene expression across different leukocyte populations in scRNAseq data from atherosclerotic LDLR-/- mice. To evaluate the role of macrophage expressed CD36 in atherogenesis, we generated tamoxifen inducible macrophage (CX3CR1)-specific CD36-deficient mice on a LDLR-/- background. To interrogate a potential role of CD36 in human atherosclerosis, human atherosclerotic plaques were studied by scRNAseq. Results: Analysis of murine scRNAseq data identified macrophages as the main CD36 expressing cell type. In line, CD36 protein expression in aortic cell suspensions from LDLR-/- mice was significantly higher in macrophages compared to B and T cells as assessed by flow cytometry. Notably, macrophage-specific CD36 deficiency substantially reduced lesional necrotic core area after 16 weeks of high cholesterol diet feeding indicating ameliorated plaque vulnerability. In vitro, macrophage-specific CD36 deficiency reduced ROS production and oxLDL uptake. Similar to our observations in mice, cellular CD36 expression was highest in human macrophages/monocytes. Pathway analysis revealed that CD36-expressing macrophages exhibited a more pro-inflammatory gene signature than CD36-negative macrophages. Finally, unstable carotid plaques displayed a significantly higher CD36 expression compared to stable lesions. Conclusion: All in all, our data indicates a pro-inflammatory role of macrophage expressed CD36 in murine and human atherosclerosis. These findings suggest that targeting CD36 on macrophages may represent a potential target for anti-atherosclerotic therapy.

Abstract 14517: apoE Receptor-2-R952Q Variant in Macrophages Potentiates Hyperlipidemia and Atherosclerosis in LDL Receptor-Knockout Mice via Increased Secretion of Soluble LDL Receptor-Related Protein-1

Introduction and objective: Apolipoprotein E Receptor-2 (apoER2) is a type I transmembrane receptor in the LDLR family 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 in humans. This study investigated the role of the R952Q mutation of apoER2 in modulating disease development. Methods and Results: We generated R952Q-expressing mice using a CRISPR-directed approach. This mouse line was crossed with the LDLR knockout mouse line and the progenies were fed a high cholesterol diet for 16 or 24 weeks. Results showed that unlike apoER2 knockout, the accelerated atherosclerosis R952Q variant mice exhibited was linked to increased plasma lipid levels. Considering apoER2's expression, which is notably absent in hepatocytes, we investigated the impact of R952Q myeloid cells on disease progression via bone marrow transplant. Results showed that wildtype apoER2 mouse recipients of R952Q bone marrow recapitulated the elevated plasma lipid level phenotype while R952Q mouse recipients of wildtype apoER2 bone marrow did not. Due to the absence of LDLR in our model, we investigated LRP1’s role in the context of myeloid-specific R952Q and found an increase in the production of hypercholesterolemia-associated soluble LRP1 (sLRP1) in the plasma of high cholesterol diet-fed mice with myeloid-specific R952Q. sLRP1 may compete with cell-surface LRP1 in lipoprotein clearance, suggesting a novel mechanism by which the R952Q mutation in macrophages may increase plasma lipid levels, thereby accelerating atherosclerosis. Conclusion: The R952Q mutation in apoER2 accelerates atherosclerosis via overproduction of soluble LRP1, a gain-of-function mechanism in macrophages resulting in hypercholesterolemia.

Abstract 17202: Cross-Species Evaluation of Cellular TNF Receptor-Associated Factor (TRAF) Expression, Regulation and Function in Murine and Human Atherosclerosis

Background: TNF receptor-associated factors (TRAFs) potently modulate immune cell function and activity in a variety of inflammatory diseases. However, due to the limited availability of viable animal models, the roles of TRAF3, TRAF4 and TRAF7 in atherosclerosis remain unknown to date. Here, we present a stepwise multiparametric approach to delineate cellular expression, regulation and implications on cardiovascular outcomes of TRAFs in murine and human atherosclerosis. Methods: To induce atherosclerotic lesion formation, LDLR-/- mice were fed a high cholesterol diet for 20 weeks and expression of TRAF1-7 was determined in whole tissue lysates of the murine aorta as well as on sorted leukocytes. We then performed scRNAseq and bioinformatical integration of leukocytes from human and mouse atherosclerotic plaques to analyze and compare single cell TRAF expression in both species. Finally, potential significance of cellular TRAF3, 4 and 7 expression in human atherosclerosis was assessed in three independent clinical study cohorts. Results: Aortic TRAF3 and TRAF4 expression was 2-fold higher in atherosclerotic mice compared to controls, whereas TRAF7 expression was significantly lower. Between species, humans exhibited higher cellular TRAF3 expression than mice. Further, TRAF7 blood expression was lower in patients with an unstable CAD angiogram than in patients with stable CAD while expression of TRAF3 in human atherosclerotic plaques was significantly higher in unstable compared to stable lesions. Finally, TRAF3 expression on circulating CD16+ monocytes determined by scRNAseq of PBMCs was substantially higher in atherosclerotic patients that died from a cardiovascular cause during 20 years of follow-up compared to survivors from the same study collective, indicating that cellular TRAF expression may predict cardiovascular long-term outcomes. Conclusion: Here, we present the first cross-species assessment of cellular TRAF3, 4 and 7 expression in murine and human atherosclerosis by utilizing a multiparametric approach to circumvent the lack of viable knockout models. Finally, we provide first evidence that cellular TRAF expression assessed by scRNAseq may add to the clinicians armamentarium for MACE prediction in the future.

Hydroxypropyl-β-cyclodextrin inhibits the development of triple negative breast cancer by enhancing antitumor immunity

Triple negative breast cancer (TNBC) is regarded as one of the most aggressive forms of breast cancer. Hydroxypropyl-β-cyclodextrin (HP-β-CD) has been used as a therapeutic agent for Niemann-Pick disease Type C (NPC). However, the exact actions and mechanisms of HP-β-CD on TNBC are not fully understood. To examine the influence of HP-β-CD on the proliferation and migration of TNBC cell lines, particularly 4T1 and MDA-MB-231 cells, a range of assays, including MTT, scratch, cell cycle, and clonal formation assays, were performed. Furthermore, the effectiveness of HP-β-CD in the treatment of TNBC was assessed in vivo using a 4T1 tumor-bearing BALB/c mouse model. We demonstrated the anti-proliferation and anti-migration effect of HP-β-CD on TNBC both in vitro and in vivo. High cholesterol diet can attenuate HP-β-CD-inhibited TNBC growth. Mechanistically, HP-β-CD reduced tumor cholesterol levels by increasing ABCA1 and ABCG1-mediated cholesterol reverse transport. HP-β-CD promoted the infiltration of T cells into the tumor microenvironment (TME) and improved exhaustion of CD8+ T cells via reducing immunological checkpoint molecules expression. Additionally, HP-β-CD inhibited the recruitment of tumor associated macrophages to the TME via reducing CCL2-p38MAPK-NF-κB axis. HP-β-CD also inhibited the epithelial mesenchymal transition (EMT) of TNBC cells mediated by the TGF-β signaling pathway. In summary, our study suggests that HP-β-CD effectively inhibited the proliferation and metastasis of TNBC, highlighting HP-β-CD may hold promise as a potential antitumor drug.

Insight to phytochemical investigation and anti-hyperlipidemic effects of Eucalyptus camaldulensis leaf extract using in vitro, in vivo and in silico approach

Hyperlipidemia is a key risk factor mainly for hypertension and cardiac abnormalities. Previously eucalyptus plant (river red gum) had been used for its medicinal value for the treatment of many ailments. This study focused on phytochemical examination, investigation of an in vitro potential and in vivo effects in mice fed with high cholesterol diet, GC-MS analysis of extracts of Eucalyptus camaldulensis leaves and further confirmation of anti-hyperlipidemic potential of different constituents of plant extracts by using in silico technique. For in vitro study screening of different extracts of Eucalyptus camaldulensis leaves was performed by using pancreatic lipase enzyme inhibition assay. Ethanolic extract presented the highest potential among all the extracts by inhibiting pancreatic lipase having IC50–11.88 µg/mL. For in vivo study mice were fed with high cholesterol diet for induction of Hyperlipidemia. Water extract showed great anti-hyperlipidemic potential by reducing the level of cholesterol, triglycerides, low density lipoproteins and increasing high density lipoproteins level significantly (p < 0.05). Moreover, molecular docking and prime MM-GBSA study were applied for screening of compounds having anti-hyperlipidemic potential which showed that Alpha-cadinol was the lead compound for inhibition of pancreatic lipase enzyme having docking score (-6.604). The ADMET properties and toxicity profile of the top docked compounds were also detailed for ensuring their safety aspects. In this way in silico analysis substantiate the experimental findings by showing anti-hyperlipidemic potential in constituents of eucalyptus plant. Thus, there is a need of advanced research for isolation of active constituents having said anti-hyperlipidemic potential in the Eucalyptus camaldulensis plant. Communicated by Ramaswamy H. Sarma

PSVI-2 Dietary Supplementation of Phyllanthus Urinaria Ameliorates Fatty Liver Hemorrhagic Syndrome in Hens

Abstract The purpose of the present study was to investigate the effect of Phyllanthus urinaria supplement on fatty liver hemorrhagic syndrome (FLHS) in hens. Forty 7-week-old ISA hens were randomly divided into four groups (n = 10), including control diet group (CON), high cholesterol and low choline diet group (CLC), and CLC with 1% P. urinaria powder group (PU). CLC diet and the supplementation of P. urinaria for six weeks exhibited no impact on feed intake and body weight (P &amp;gt; 0.05). The triglyceride (TG) and the total cholesterol (TC) in plasma was increased at week 6 (TG: CON = 18.86 ± 0.97 mg/dL and CLC = 66.45 ± 7.17 mg/dL; TC: CON = 130.41 ± 5.87 mg/dL and CLC = 308.11 ± 27.97 mg/dL); nevertheless, P. urinaria decreased the triglyceride in plasma (PU = 41.19 ± 4.88 mg/dl, P &amp;lt; 0.05), but not total cholesterol (PU = 259.11 ± 20.88 mg/dL, P &amp;gt; 0.05). The malondialdehyde (MDA) concentration in plasma was decreased with 1% P. urinaria powder group (CLC = 64.81 ± 3.71 mg/dL and PU = 49.13 ± 4.20 mg/dL, P &amp;lt; 0.05). Under liver macroscopic analysis, PU demonstrated the least percentage of severe in hepatic appearance (CLC = 22% and PU = 0%). Similarly, the liver microscopic examination (H&amp;E) suggested the PU supplementation reduced steatosis score (CLC = 2.89 ± 0.11 and PU = 2.11 ± 0.16) and blooning (CLC = 1.63 ± 0.22 and PU = 0.88 ± 0.31) in the hepatocyte. To sum up, this study revealed the hepatoprotective effect of Phyllanthus urinaria and provided a potential strategy for preventing FLHS to improve the welfare in hens.