Accumulation Of Low-density Lipoprotein Research Articles

IL-1β Induces LDL Transcytosis by a Novel Pathway Involving LDLR and Rab27a.

In early atherosclerosis, circulating LDLs (low-density lipoproteins) traverse individual endothelial cells by an active process termed transcytosis. The CANTOS trial treated advanced atherosclerosis using a blocking antibody for IL-1β (interleukin-1β); this significantly reduced cardiovascular events. However, whether IL-1β regulates early disease, particularly LDL transcytosis, remains unknown. We used total internal reflection fluorescence microscopy to quantify transcytosis by human coronary artery endothelial cells exposed to IL-1β. To investigate transcytosis in vivo, we injected wild-type and knockout mice with IL-1β and LDL to visualize acute LDL deposition in the aortic arch. Exposure to picomolar concentrations of IL-1β induced transcytosis of LDL but not of albumin by human coronary artery endothelial cells. Surprisingly, expression of the 2 known receptors for LDL transcytosis, ALK-1 (activin receptor-like kinase-1) and SR-BI (scavenger receptor BI), was unchanged or decreased. Instead, IL-1β increased the expression of the LDLR (LDL receptor); this was unexpected because LDLR is not required for LDL transcytosis. Overexpression of LDLR had no effect on basal LDL transcytosis. However, knockdown of LDLR abrogated the effect of IL-1β on transcytosis rates while the depletion of Cav-1 (caveolin-1) did not. Since LDLR was necessary but overexpression had no effect, we reasoned that another player must be involved. Using public RNAseq data to curate a list of Rab GTPases affected by IL-1β, we identified Rab27a. Overexpression of Rab27a alone had no effect on basal transcytosis, but its knockdown prevented induction by IL-1β. This was phenocopied by depletion of the Rab27a effector JFC1. In vivo, IL-1β increased LDL transcytosis in the aortic arch of wild-type but not Ldlr-/- or Rab27a-deficient mice. The JFC1 inhibitor nexinhib20 also blocked IL-1β-induced LDL accumulation in the aorta. IL-1β induces LDL transcytosis by a distinct pathway requiring LDLR and Rab27a; this route differs from basal transcytosis. We speculate that induction of transcytosis by IL-1β may contribute to the acceleration of early disease.

Aptamer and sodium alginate decorated graphene oxide composite material with ion responsiveness for Low-density lipoprotein trapping

The accumulation of excess Low-density lipoprotein (LDL) is strongly associated with the occurrence of heart failure, coronary artery disease and hypercholesterolaemia, and is a major factor in cardiovascular and cerebrovascular disease. Concerns about the ways to decrease LDL level have continuously arisen. In this study, an ionic stimulation-responsive composite (i.e., GO@Apt@SA) is prepared with modification of graphene oxide (GO) utilising LDL-aptamer (Apt) and sodium alginate (SA). The ion-responsive behaviour of GO@Apt@SA synergistically interacts with the specific recognition property of the aptamer, enabling adsorption of LDL with higher capacity and specificity. Under the optimal experimental conditions, the maximum adsorption capacity of GO@Apt@SA for LDL is 730.6 μg mg−1. Interestingly, the aptamer complementary chain could trigger the release of LDL with favourable elution efficiency, which competitively binds with LDL-specific aptamer to trigger LDL release. More importantly, GO@Apt@SA exhibits satisfactory adsorption performances for LDL in goat serum, meaning that the composite material and technology are available for the extraction of LDL from complex sample matrices.

Simultaneous Rosiglitazone Release and Low-Density Lipoprotein Removal by Chondroitin Sodium Sulfate/Cyclodextrin/Poly(acrylic acid) Composite Adsorbents for Atherosclerosis Therapy.

Atherosclerosis (AS) is characterized by the accumulation of substantial low-density lipoprotein (LDL) and inflammatory response. Hemoperfusion is commonly employed for the selective removal of LDL from the body. However, conventional hemoperfusion merely focuses on LDL removal and does not address the symptom of plaque associated with AS. Based on the LDL binding properties of acrylated chondroitin sodium sulfate (CSA), acrylated beta-cyclodextrin (CD) and acrylic acid (AA), along with the anti-inflammatory property of rosiglitazone (R), the fabricated AA-CSA-CD-R microspheres could simultaneously release R and facilitate LDL removal for hemoperfusion. The AA and CSA offer electrostatic adsorption sites for LDL, while the CD provides hydrophobic adsorption sites for LDL and weak binding sites for R. According to the Sips model, the maximum static LDL adsorption capacity of AA-CSA-CD-R is determined to be 614.73 mg/g. In dynamic simulated perfusion experiments, AA-CSA-CD-R exhibits an initial cycle LDL adsorption capacity of 150.97 mg/g. The study suggests that the weakened inflammatory response favors plaque stabilization. The anti-inflammatory property of the microspheres is verified through an inflammation model, wherein the microsphere extracts are cocultured with mouse macrophages. Both qualitative analysis of iNOS\TNF-α and quantitative analysis of IL-6\TNF-α collectively demonstrate the remarkable anti-inflammatory effect of the microspheres. Therefore, the current study presents a novel blood purification treatment of eliminating pathogenic factors and introducing therapeutic factors to stabilize AS plaque.

Oxidized Low-Density Lipoprotein Accumulation in Macrophages Impairs Lipopolysaccharide-Induced Activation of AKT2, ATP Citrate Lyase, Acetyl-Coenzyme A Production, and Inflammatory Gene H3K27 Acetylation.

The accumulation of lipid and the formation of macrophage foam cells is a hallmark of atherosclerosis, a chronic inflammatory disease. To better understand the role of macrophage lipid accumulation in inflammation during atherogenesis, we studied early molecular events that follow the accumulation of oxidized low-density lipoprotein (oxLDL) in cultured mouse macrophages. We previously showed that oxLDL accumulation downregulates the inflammatory response in conjunction with downregulation of late-phase glycolysis. In this study, we show that within hours after LPS stimulation, macrophages with accumulated oxLDL maintain early-phase glycolysis but selectively downregulate activation of AKT2, one of three AKT isoforms. The inhibition of AKT2 activation reduced LPS-induced ATP citrate lyase activation, acetyl-CoA production, and acetylation of histone 3 lysine 27 (H3K27ac) in certain inflammatory gene promoters. In contrast to oxLDL, multiple early LPS-induced signaling pathways were inhibited in macrophages with accumulated cholesterol, including TBK1, AKT1, AKT2, MAPK, and NF-κB, and early-phase glycolysis. The selective inhibition of LPS-induced AKT2 activation was dependent on the generation of mitochondrial oxygen radicals during the accumulation of oxLDL in macrophages prior to LPS stimulation. This is consistent with increased oxidative phosphorylation, fatty acid synthesis, and oxidation pathways found by comparative transcriptomic analyses of oxLDL-loaded versus control macrophages. Our study shows a functional connection between oxLDL accumulation, inactivation of AKT2, and the inhibition of certain inflammatory genes through epigenetic changes that occur soon after LPS stimulation, independent of early-phase glycolysis.

Investigating SHP and PCSK9 Interactions in Cholesterol-Mediated Cardiovascular Diseases: A Research Protocol

Introduction: Improper cholesterol metabolism results in accumulation of low-density lipoproteins (LDL). High levels of LDL cholesterol deposits in blood vessels, forming plaques and contributing to various cardiovascular diseases (CVD). The nuclear farnesoid X receptor (FXR) regulates the transcription of genes involved in cholesterol metabolism and is a therapeutic target for cholesterol dysregulation. Studies conducted on immortalized human hepatocytes demonstrate FXR signaling-induced downregulation of proprotein convertase subtilisin/kexin type 9 (PCSK9) expression. PCSK9 is an LDL receptor-degrading enzyme whose upregulation is implicated in cholesterol-mediated diseases. Specifically, the FXR target gene SHP (small heterodimer partner) is a transcriptional regulator that has been implicated in an inverse relationship with PCSK9 expression. The biomolecular mechanism mediating this relationship has not been explored, meriting investigation into a potential novel axis of cholesterol metabolism. We hypothesize that SHP is a direct repressor of PCSK9 transcription. Methods: To investigate, we will knock out SHP expression in the liver hepatocyte cell line AML12 using small interfering RNAs (siRNAs). To confirm SHP knockout on transcriptomic and proteomic levels, reverse transcription quantitative PCR (RT-qPCR) and Western blotting will be performed. To assess SHP binding to the promoter region of PCSK9, an electrophoretic mobility supershift (EMSA) assay will be performed on unstimulated or chenodeoxycolic acid (CDCA)-stimulated AML12 cells that have undergone SHP or control knockouts. Western blotting will quantitate PCSK9 protein expression following SHP knockout in CDCA-stimulated and unstimulated conditions. Results: Results from EMSA are expected to demonstrate SHP binding to the promoter region of PCSK9 in a transcription factor complex to repress transcription. SHP knockout models are expected to show upregulated PCSK9 expression at transcriptomic and proteomic levels. Discussion: If successful, our study presents a novel perspective on cholesterol metabolism by characterizing the inhibitory effect of SHP on PCSK9 expression. This underlines the critical role of FXR signaling in PCSK9 regulation, and knockout models and assay techniques provide valuable evidence of this regulatory role. Conclusion: This study will establish an enhanced understanding of the SHP/PCSK9 pathway within broader pathways of cholesterol metabolism. Further research may explore therapies targeting the SHP/PCSK9 pathway to manage CVD downstream of cholesterol dysregulation.

The Link between miRNAs and PCKS9 in Atherosclerosis.

Cardiovascular disease (CDV) represents the major cause of death globally. Atherosclerosis, as the primary cause of CVD, is a chronic immune-inflammatory disorder with complex multifactorial pathophysiology encompassing oxidative stress, enhanced immune-inflammatory cascade, endothelial dysfunction, and thrombosis. An initiating event in atherosclerosis is the subendothelial accumulation of low-density lipoprotein (LDL), followed by the localization of macrophages to fatty deposits on blood vessel walls, forming lipid-laden macrophages (foam cells) that secrete compounds involved in plaque formation. Given the fact that foam cells are one of the key culprits that underlie the pathophysiology of atherosclerosis, special attention has been paid to the investigation of the efficient therapeutic approach to overcome the dysregulation of metabolism of cholesterol in macrophages, decrease the foam cell formation and/or to force its degradation. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory serine proteinase that has emerged as a significant regulator of the lipid metabolism pathway. PCSK9 activation leads to the degradation of LDL receptors (LDLRs), increasing LDL cholesterol (LDL-C) levels in the circulation. PCSK9 pathway dysregulation has been identified as one of the mechanisms involved in atherosclerosis. In addition, microRNAs (miRNAs) are investigated as important epigenetic factors in the pathophysiology of atherosclerosis and dysregulation of lipid metabolism. This review article summarizes the recent findings connecting the role of PCSK9 in atherosclerosis and the involvement of various miRNAs in regulating the expression of PCSK9-related genes. We also discuss PCSK9 pathway-targeting therapeutic interventions based on PCSK9 inhibition, miRNA levels manipulation by therapeutic agents, and the most recent advances in PSCK9 gene editing using CRISPR/Cas9 platform, meganuclease, and base editors.

PCSK9 Inhibitor Therapy as an Alternative for Statin Intolerance

Scientific relevance. The main cause of cardiovascular pathologies is atherosclerosis, which is secondary to lipid metabolism disorders, in particular, the accumulation of low-density lipoprotein (LDL) cholesterol. Dyslipidaemia treatment with the largest evidence base predominantly includes statins in combination therapy, but their use is limited by into lerance in some patients. Alternatively, the treatment may include proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors.Aim. The study aimed to analyse the applicability of PCSK9 inhibitors in patients with statin intolerance.Discussion. According to the literature analysis, the most common presentation of statin intolerance is statin-associated muscle symptoms. The pathogenesis of statin-associated adverse events is mainly mediated by HMGCoA reductase inhibition, treatment effects on cellular and subcellular processes and skeletal muscles, and patients’ genetic makeup. The mechanism of action of PCSK9 inhibitors is entirely different and involves binding and inactivation of the PCSK9 protein, which lowers blood LDL cholesterol levels. PCSK9 inhibitors have been associated with some adverse drug reactions, most notably immunogenicity; however, PCSK9 inhibitors effectively reduce LDL levels even if patients develop antibodies.Conclusions. Therefore, PCSK9 inhibitors are a safe, well-tolerated, and effec tive therapeutic strategy for hyperlipidaemia in patients with statin intolerance.

Inflammatory Cell-Derived MYDGF Attenuates Endothelial LDL Transcytosis to Protect Against Atherogenesis.

Inflammation contributes to the pathogenesis of atherosclerosis. But little is known about the potential benefits of inflammatory cells to atherosclerosis. The aim of this study was to investigate the function of inflammatory cells/endothelium axis and determine whether and how inflammatory cell-derived MYDGF (myeloid-derived growth factor) inhibited endothelial LDL (low-density lipoprotein) transcytosis. In in vivo experiments, both loss- and gain-of-function strategies were used to evaluate the effect of inflammatory cell-derived MYDGF on LDL transcytosis. We generated monocyte/macrophage-targeted MYDGF-null mice on an Ldlr (LDL receptor)-/- background in the loss-of-function strategy and restored the inflammatory cell-derived MYDGF by bone marrow transplantation and inflammatory cell-specific overexpression of MYDGF mice model in the gain-of-function strategy. In in vitro experiments, coculture experiments between primary mouse aortic endothelial cells and macrophages and mouse aortic endothelial cells supplemented with or without recombinant MYDGF were conducted. Inflammatory cell-derived MYDGF deficiency aggravated endothelial LDL transcytosis, drove LDL uptake by artery wall, and thus exacerbated atherosclerosis in vivo. Inflammatory cell-derived MYDGF restoration by bone marrow transplantation and inflammatory cell MYDGF overexpression alleviated LDL transport across the endothelium, prevented LDL accumulation in the subendothelial space, and subsequently ameliorated atherosclerosis in vivo. Furthermore, in the in vitro study, macrophages isolated from MYDGF+/+ mice and recombinant MYDGF attenuated LDL transcytosis and uptake in mouse aortic endothelial cells. Mechanistically, MYDGF inhibited MAP4K4 (mitogen-activated protein kinase kinase kinase kinase isoform 4) phosphorylation, enhanced activation of Akt (protein kinase B)-1, and diminished the FoxO (forkhead box O) 3a signaling cascade to exert protective effects of MYDGF on LDL transcytosis and atherosclerosis. The findings support a role for inflammatory cell-derived MYDGF served as a cross talk factor between inflammatory cells and endothelial cells that inhibits LDL transcytosis across endothelium. MYDGF may become a novel therapeutic drug for atherosclerosis, and the beneficial effects of inflammatory cell in atherosclerosis deserve further attention.

Oxidized Low-Density Lipoprotein Accumulation Suppresses Glycolysis and Attenuates the Macrophage Inflammatory Response by Diverting Transcription from the HIF-1α to the Nrf2 Pathway.

Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild-type mouse thioglycolate-elicited peritoneal Mφs and bone marrow-derived Mφs with conditional (Lyz2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2, and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 h with oxidized low-density lipoprotein (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs downregulated inflammatory, hypoxia, and cholesterol metabolism pathways, whereas the antioxidant pathway, fatty acid oxidation, and ABC family proteins were upregulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both hypoxia-inducible factor 1-α (HIF-1α) stability and transactivation capacity; thus, the phenotype was not rescued in Vhl-/- Mφs. Intracellular lipid accumulation in Mφs also enhanced LPS-induced NF erythroid 2-related factor 2 (Nrf2)-mediated antioxidative defense that destabilizes HIF-1α, and Nrf2-deficient Mφs resisted the inhibitory effects of lipid accumulation on glycolysis and inflammatory gene expression. Furthermore, oxLDL shifted NADPH consumption from HIF-1α- to Nrf2-regulated apoenzymes. Thus, we postulate that repurposing NADPH consumption from HIF-1α to Nrf2 transcriptional pathways is critical in modulating inflammatory responses in Mφs with accumulated intracellular lipid. The relevance of our invitro models was established by comparative transcriptomic analyses, which revealed that Mφs cultured with oxLDL and stimulated with LPS shared similar inflammatory and metabolic profiles with foamy Mφs derived from the atherosclerotic mouse and human aorta.

Computational modeling of low-density lipoprotein accumulation at the carotid artery bifurcation after stenting.

Restenosis typically occurs in regions of low and oscillating wall shear stress, which also favor the accumulation of atherogenic macromolecules such as low-density lipoprotein (LDL). This study aims to evaluate LDL transport and accumulation at the carotid artery bifurcation following carotid artery stenting (CAS) by means of computational simulation. The computational model consists of coupled blood flow and LDL transport, with the latter being modeled as a dilute substance dissolved in the blood and transported by the flow through a convection-diffusion transport equation. The endothelial layer was assumed to be permeable to LDL, and the hydraulic conductivity of LDL was shear-dependent. Anatomically realistic geometric models of the carotid bifurcation were built based on pre- and post-stent computed tomography (CT) scans. The influence of stent design was investigated by virtually deploying two different types of stents (open- and closed-cell stents) into the same carotid bifurcation model. Predicted LDL concentrations were compared between the post-stent carotid models and the relatively normal contralateral model reconstructed from patient-specific CT images. Our results show elevated LDL concentration in the distal section of the stent in all post-stent models, where LDL concentration is 20 times higher than that in the contralateral carotid. Compared with the open-cell stents, the closed-cell stents have larger areas exposed to high LDL concentration, suggesting an increased risk of stent restenosis. This computational approach is readily applicable to multiple patient studies and, once fully validated against follow-up data, it can help elucidate the role of stent strut design in the development of in-stent restenosis after CAS.

Targeting structural flexibility in low density lipoprotein by integrating cryo-electron microscopy and high-speed atomic force microscopy

Low-density lipoprotein (LDL) plays a crucial role in cholesterol metabolism. Responsible for cholesterol transport from the liver to the organs, LDL accumulation in the arteries is a primary cause of cardiovascular diseases, such as atherosclerosis. This work focuses on the fundamental question of the LDL molecular structure, as well as the topology and molecular motions of apolipoprotein B-100 (apo B-100), which is addressed by single-particle cryo-electron microscopy (cryo-EM) and high-speed atomic force microscopy (HS-AFM). Our results suggest a revised model of the LDL core organization with respect to the cholesterol ester (CE) arrangement. In addition, a high-density region close to the flattened poles could be identified, likely enriched in free cholesterol. The most remarkable new details are two protrusions on the LDL surface, attributed to the protein apo B-100. HS-AFM adds the dimension of time and reveals for the first time a highly dynamic direct description of LDL, where we could follow large domain fluctuations of the protrusions in real time. To tackle the inherent flexibility and heterogeneity of LDL, the cryo-EM maps are further assessed by 3D variability analysis. Our study gives a detailed explanation how to approach the intrinsic flexibility of a complex system comprising lipids and protein.

Novel soybean polypeptide dglycin alleviates atherosclerosis in apolipoprotein E-deficient mice

Atherosclerosis is a dominant cause of cardiovascular disease. Accumulation of low-density lipoproteins (LDL), formation of foam cells, and endothelial dysfunction within the arterial intima contribute to atherosclerotic plaque formation. Soy consumption is thought to have positive effect on the prevention of atherosclerosis. Therefore, in the present study, a novel soybean polypeptide dglycin was purified and characterized. Oral administration of 20 mg/g.d dglycin reduced 47.6 % lesion area, and 49.1 % lipid deposition in the atherosclerotic plaques in aortic roots in ApoE−/− mice. In addition, it decreased the levels of 26.0 % plasma low-density lipoprotein, 27.2 % triglyceride, 40.1 % cholesterol, 25.1 % malondialdehyde and 24.2 % tumor necrosis factor-alpha (TNFα). In vitro experiments revealed that dglycin inhibited inflammatory cytokine secretion from aortic endothelial cells via the inhibition of NF-κB signaling. Furthermore, it inhibited reactive oxygen species generation, subsequently enhanced cell viability, and protected aortic endothelial cells from necrosis and apoptosis via mitochondrial function improvement. On the other hand, dglycin prevented the uptake of oxidized LDL by macrophages via suppressing the expression of scavenger receptor class A1, which suggested that dglycin prevented foam cell formation. Therefore, dglycin alleviated the early-stage of atherosclerosis via depressing inflammation, lipid deposition, protecting aortic endothelial cells and preventing foam cell formation.

Benzo[a]pyrene and a high-fat diet induce aortic injury and promote low-density lipoprotein accumulation in the endothelium

Benzo[a]pyrene (BaP) is a ubiquitous environmental pollutant which mainly exposed though diet. High-fat diet (HFD) can induce atherosclerosis, as can BaP. Unhealthy dietary habits lead to high intake of both BaP and lipids. However, the combined effect of BaP and HFD on atherosclerosis and lipid accumulation in the arterial wall, the initial stage of atherosclerosis, is unclear. In this study, C57BL/6 J mice were subchronically exposed to BaP and a HFD, and the mechanism of lipid accumulation was investigated in EA.hy926 and HEK293 cells. Results showed that BaP and HFD increased blood lipids and damaged aortic wall synergistically. Meanwhile, LDL enhanced the toxicity of BaP, and BaP promoted the production of reactive oxygen species and malonaldehyde in EA.hy926 cells, which aggravated LDL-induced cell injury. Moreover, BaP and HFD/LDL induced LDL accumulation in the aortic wall of C57BL/6 J mice/EA.hy926, and the mechanism was by activating AHR/ARNT heterodimer to combine with the scavenger receptor BⅠ (SR-BⅠ) and activin receptor-like kinase 1 (ALK1) promoter regions to transcriptional upregulate its expression, which enhanced the uptake of LDL, and promoting the production of AGEs to inhibit reverse cholesterol transport by SR-BI. BaP and lipid synergistically promoted aortic and endothelial damage, and the health risk of their combined intake should be paid attention to.

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.

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

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

Liver lipidomics analysis reveals the anti-obesity and lipid-lowering effects of gypnosides from heat-processed Gynostemma pentaphyllum in high-fat diet fed mice

BackgroundIn traditional Chinese medicine, Gynostemma pentaphyllum (G. pentaphyllum) is widely used to treat conditions associated with hyperlipidemia, and its therapeutic potential has been demonstrated in numerous studies. However, the mechanism of lipid metabolism in hyperlipidemic by G. pentaphyllum, especially heat-processed G. pentaphyllum is not yet clear. PurposeThe aim of this study was to investigate the therapeutic mechanism of gypenosides from heat-processed G. pentaphyllum (HGyp) in hyperlipidemic mice by means of a lipidomics. MethodsThe content of the major components of HGyp was determined by ultra-performance liquid chromatography-electrospray ionization ion trap mass spectrometry (UPLC-ESI-MS). An animal model of hyperlipidaemia was constructed using C57BL/6J mice fed with high-fat diet. HGyp was also administered at doses of 50, 100 and 200 mg/kg, all for 12 weeks. Serum parameters were measured, histological sections were prepared and liver lipidome analysis using UPLC-MS coupled with multivariate statistical analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to analyze the genes and proteins associated with lipid lowering in HGyp. ResultsHGyp reduced body weight, serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL) and hepatic lipid accumulation in hyperlipidemic obese mice. To explore specific changes in lipid metabolism in relation to HGyp administration, lipid analysis of the liver was performed. Orthogonal partial least squares discriminant analysis (OPLS-DA) score plots showed that HGyp altered lipid metabolism in HFD mice. In particular, fatty acids (FA), triglycerides (DG), TG and ceramides (CER) were significantly altered. Eleven lipids were identified as potential lipid biomarkers, namely TG (18:2/20:5/18:2), TG (18:2/18:3/20:4), DG (18:3/20:0/0:0), Cer (d18:1/19:0), Cer (d16:1/23:0), Ceramide (d18:1/9Z-18:1), PS (19:0/18:3), PS (20:2/0:0), LysoPC (22:5), LysoPE (0:0/18:0), PE (24:0/16:1). Western blot and qRT-PCR analysis showed that these metabolic improvements played a role by down-regulating genes and proteins related to fat production (SREBP1, ACC1, SCD1), up-regulating genes and proteins related to lipid oxidation (CPTA1, PPARα) and lipid transport decomposition in the bile acid pathway (LXRα, PPARγ, FXR, BSEP). ConclusionThe lipid-lowering effect of gypenosides from heat-processed G. pentaphyllum is regulate lipid homeostasis and metabolism.

Capacity for LDL (Low-Density Lipoprotein) Retention Predicts the Course of Atherogenesis in the Murine Aortic Arch.

To cause atherosclerosis, LDLs (low-density lipoproteins) must first pass through the endothelium and then become retained in the arterial matrix. Which of these two processes is rate-limiting and predicts the topography of plaque formation remains controversial. To investigate this issue, we performed high-resolution mapping of LDL entry and retention in murine aortic arches before and during atherosclerosis development. Maps of LDL entry and retention were created by injecting fluorescently labeled LDL followed by near-infrared scanning and whole-mount confocal microscopy after 1 hour (entry) and 18 hours (retention). By comparing arches between normal mice and mice with short-term hypercholesterolemia, we analyzed changes in LDL entry and retention during the LDL accumulation phase that precedes plaque formation. Experiments were designed to secure equal plasma clearance of labeled LDL in both conditions. We found that LDL retention is the overall limiting factor for LDL accumulation but that the capacity for LDL retention varied substantially over surprisingly short distances. The inner curvature region, previously considered a homogenous atherosclerosis-prone region, consisted of dorsal and ventral zones with high capacity and a central zone with low capacity for continued LDL retention. These features predicted the temporal pattern of atherosclerosis, which first appeared in the border zones and later in the central zone. The limit to LDL retention in the central zone was intrinsic to the arterial wall, possibly caused by saturation of the binding mechanism, and was lost upon conversion to atherosclerotic lesions. Capacity for continued LDL retention varies over short distances and predicts where and when atherosclerosis develops in the mouse aortic arch.

S-25-4: HIGHER URINARY ANGIOTENSIN CONVERTING ENZYME ACTIVITY IS OBSERVED IN BRAZILIAN CHILDREN AND ADOLESCENTS WITH DYSLIPIDEMIA

Angiotensin converting enzyme (ACE) is upregulated in patients with hyperlipidemia and atherosclerosis and contributes to oxidation and accumulation of low-density lipoproteins in blood vessels. Blockage of dyslipidemia and ACE have a synergic effect preventing or ameliorating cardiovascular disease. Objective: Investigate whether ACE activity is modulated in childhood dyslipidemia and can be associated with high cardiovascular risk profile. Design and methods: a cross-sectional study with 360 individuals (190 boys and 170 girls) aged from 6 to 19 year (mean 11.6) was performed. Anthropometric measurements, blood pressure assessment and fasting lipid concentrations were evaluated. Participants were divided according to the levels of each lipoprotein (Total cholesterol (TC), triglycerides (TG), LDL-C, HDL-C, and non-HDL) into three groups: normolipidemic, borderline and dyslipidemic. Reference values were according to American Academy of Pediatrics and Brazilian Pediatric Society. ACE activity in urine was measured with the fluorogenic substrates Z-Phe-His-Leu-OH (Z-FHL) and hippuryl-His-Leu-OH (h-HL). ACE activity ratio to infer N-domain activity was calculated (Z-FHL/h-HL). Results: Dyslipidemic levels of HDL-C, TG and LDL-C were observed in 23%, 9% and 3% of the participants, respectively. These clinical alterations were more frequent in obese children (Chi-square, p < 0.001). ACE activity measured with Z-FHL was higher in the groups with borderline and dyslipidemic values of HDL-C than in normolipidemic group (0.077 vs 0.070 vs 0.037 nmol/min/mg of creatinine, p = 0.01). The ratio of ACE activity was augmented in HDL-C borderline group when compared to HDL-C normolipidemic group (5.06 vs 2.39, p < 0.01) and in LDL-C dyslipidemic group than in LDL-C borderline and LDL-C normolipidemic groups (8.66 vs 1.84 vs 2.88, p = 0.02). Additionally, volunteers with normal levels of TG presented lower diastolic blood pressure (DBP) mmHg (p = 0.02), percentile of DBP (p < 0.01), and percentile of systolic blood pressure (SBP) (p < 0.01) than volunteers with borderline and dyslipidemic levels of TG. Also, increased DBP mmHg was observed in borderline and dyslipidemic HDL-C groups when compared to normolipidic HDL-C group. Conclusion: ACE presented a cross-link with dyslipidemia in childhood and adolescence, as hypertriglyceridemia, HDL-C deficiency and high LDL-C were related to higher ACE activity (mainly N-domain able to inactivate angiotensin 1–7, a vasodilator peptide). ACE upregulation, dyslipidemia and obesity are known to activate molecular mechanism contributing to high cardiovascular risk profile, in agreement, impairment in lipoprotein levels was associated with higher blood pressure. This is relevant evidence that ACE modulation can contribute to higher risk of hypertension and other cardiometabolic alterations still in childhood and adolescence.

Effects of Fermented Citrus Peel on Ameliorating Obesity in Rats Fed with High-Fat Diet.

Although citrus peel is a waste material, it contains a variety of bioactive components. As our preliminary findings showed that citrus peels fermented with Saccharomyces cerevisiae T1 contained increased levels of anti-obesity flavonoids, the objective of this study was to prepare fermented citrus peel and to investigate its effect on ameliorating obesity in Sprague Dawley (SD) rats fed with a high-fat diet (HFD). After fermentation, the amounts of limonene, nobiletin and 3-methoxynobiletin in citrus peel were markedly increased. SD rats were fed with an HFD for 10 weeks, followed by fermented citrus peel-containing HFD (0.3% or 0.9% w/w) for 6 weeks. Compared with those fed with an HFD alone, lower levels of body weight, visceral fat, body fat percentage, blood triglyceride, total cholesterol, low-density lipoprotein, malondialdehyde and hepatic adipose accumulation were observed in rats fed with fermented citrus peel. In parallel, hepatic levels of acetyl-CoA carboxylase and fatty acid synthase were diminished, and the level of hormone sensitivity lipase in visceral fat was elevated. These results reveal fermented citrus peel is a promising natural product with beneficial effects of alleviating HFD-induced obesity.

Simulation of LDL permeation into multilayer wall of a coronary bifurcation using WSS-dependent model: effects of hemorheology.

Atherosclerosis, due to the permeation of low-density lipoprotein (LDL) particles into the arterial wall, is one of the most common and deadly diseases in today's world. Due to its importance, numerous studies have been conducted on the factors affecting this disease. In this study, using numerical simulation, the effects of Wall Shear Stress (WSS), non-Newtonian behavior of blood, different values of hematocrit and blood pressure on LDL permeation into the arterial wall layers are investigated in a 4-layer wall model of a coronary bifurcation. To obtain the velocity and concentration fields in the fluid domain, the Navier-Stokes, Brinkman, and mass transfer equations are numerically solved in the lumen and wall layers. Results show that it is important to consider the effects of WSS on transport properties of endothelium layer in bifurcations and this leads to completely different concentration profiles compared to the constant properties model. Our computations show that a giant accumulation of LDL in the intima layer of the outer wall of the left anterior descending artery, especially in low WSS regions, may lead to atherosclerosis. It is also, necessary to consider the non-Newtonian behavior of blood in bifurcations due to its direct effect on WSS. A pressure-induced increase in the half-width of leaky junctions may be responsible for the higher risk of atherosclerosis in hypertension. In addition, it is shown that the dominant mechanism in LDL permeation into the wall is convection, and also, hypertension increases the effect of mass transfer by convection mechanism more than the diffusion mechanism. Furthermore, our results are consistent with various clinical studies.