Low-density Lipoprotein Uptake Research Articles

ECH 1 attenuates atherosclerosis by reducing macrophage infiltration and improving plaque stability through CD36 degradation

Enoyl coenzyme A hydratase 1 (ECH1) is a secreted protein implicated in numerous metabolic disorders, yet its role in the pathogenesis of atherosclerosis remains unclear. In this study, we found higher serum ECH1 levels in coronary artery disease (CAD) patients and apolipoprotein E (ApoE)−/− mice on a western diet for 12 weeks. In vivo, aorta and aortic sinus histological staining revealed that intraperitoneal injection of recombinant ECH1 reduced aortic lesions, inflammation, and macrophage infiltration in ApoE−/− mice. In vitro, incubating peritoneal macrophages with recombinant ECH1 protein reduced oxidized low-density lipoprotein uptake and increased macrophage migration. Mechanically, we observed that recombinant ECH1 incubation led to a reduction in the protein levels of scavenger receptor cluster of differentiation 36 (CD36) in primary macrophages through the promotion of CD36 protein degradation. Additionally, we found that chloroquine (CQ), a lysosomal inhibitor, mitigated this pro-degradation effect. Taken together, our findings provide unique evidence that ECH1 can attenuate the severity of atherosclerotic plaques, especially improving the stability of plaques, by decreasing macrophage infiltration. ECH1 demonstrates its protective effect by enhancing the lysosome-dependent degradation of CD36, suggesting its potential as a viable target for the prevention and treatment of atherosclerosis.

Abstract 4145859: Clonal Hematopoiesis of Indeterminate Potential Increases Mortality Risk in Coronary Artery Disease

Background: Clonal hematopoiesis of indeterminate potential (CHIP) has been associated with an increased incidence of common late-onset diseases including myocardial infarction. Research Question: Here we examined the impact of CHIP on mortality in patients with coronary artery disease (CAD) and further explored potential underlying mechanisms. Methods: CAD patients were examined for CHIP-defining mutations using deep DNA sequencing. Propensity score matching was used to compare CHIP CAD with non-CHIP CAD patients. Survival was analyzed using Kaplan-Meier curves and Cox proportional hazard models. Functional aspects were investigated at the protein and RNA level in cardiovascular relevant human postmortem tissue from the Munich cardIovaScular StudIes biObaNk (MISSION), in CAD patients from an independent cohort and in in-vitro macrophage models. Results: Within 7,933 CAD patients screened by deep-DNA-sequencing with a 13 gene panel, 2,002 carried at least one CHIP defining mutation with a variant allele frequency ≥2%. After 1:1 matching with non-CHIP carriers we revealed a significantly elevated 3-year mortality in the group of CHIP carriers [HR 1.48 (CI 95% 1.23-1.78); p<0.0001)] as well as in carriers of mutations in one out of seven individual genes – ASXL1 , DNMT3A , PPM1D , SF3B1 , SRSF2 , TET2 , and U2AF1 . In human coronary atherosclerotic plaque we visualized CHIP-mutations within leukocytes. Further, within coronary artery plaque of deceased TET2 mutation carriers (n=26 vs. n=13 controls) we identified by proteomic profiling an upregulation of inflammatory and metabolic pathways, including enhanced lipid metabolism. In an independent cohort of CAD patients undergoing coronary bypass surgery, RNA sequencing data from monocyte-derived macrophages revealed in TET2 CHIP carriers associations with CAD complexity. TET2 -mutated macrophages studied in-vitro revealed an increased uptake of low-density lipoprotein (LDL) and oxidized LDL-cholesterol besides a known upregulation of inflammatory pathways. Conclusion: In summary, this study emphasizes CHIP as a significant prognostic factor in CAD patients, associated with increased complexity of CAD and altered gene expression within human coronary artery plaques. Mechanistically, TET2 mutations modify the behavior of CHIP-affected macrophages, leading to heightened inflammation and increased uptake of LDL-cholesterol.

Piezo1-mediated mechanotransduction enhances macrophage oxidized low-density lipoprotein uptake and atherogenesis.

Macrophages in the vascular wall ingest and clear lipids, but abundant lipid accumulation leads to foam cell formation and atherosclerosis, a pathological condition often characterized by tissue stiffening. While the role of biochemical stimuli in the modulation of macrophage function is well studied, the role of biophysical cues and the molecules involved in mechanosensation are less well understood. Here, we use genetic and pharmacological tools to show extracellular oxidized low-density lipoproteins (oxLDLs) stimulate Ca2+ signaling through activation of the mechanically gated ion channel Piezo1. Moreover, macrophage Piezo1 expression is critical in the transduction of environmental stiffness and channel deletion suppresses, whereas a gain-of-function mutation exacerbates oxLDL uptake. Additionally, we find that depletion of myeloid Piezo1 protects from atherosclerotic plaque formation in vivo. Together, our study highlights an important role for Piezo1 and its respective mutations in macrophage mechanosensing, lipid uptake, and cardiovascular disease.

Nonalcoholic Fatty Liver Disease Risk and Proprotein Convertase Subtilisin Kexin 9 in Familial Hypercholesterolemia Under Statin Treatment.

Fatty acids are involved in some hepatic disorders. The proprotein convertase subtilisin kexin 9 (PCSK9) inhibits the uptake of low-density lipoproteins (LDLs), which contain lipids, into the liver and may thus be associated with nonalcoholic fatty liver disease (NAFLD), a cardiovascular disorder (CVD) risk. Statins reduce blood LDL-cholesterol (LDL-C) levels and CVD risk and can attenuate the development of NAFLD while increasing blood PCSK9 levels. We investigated the correlation between PCSK9 and liver conditions in patients with familial hypercholesterolemia (FH), a CVD risk population with elevated blood LDL-C levels, under statin treatment. Blood tests for lipids, PCSK9, and liver function (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) were performed in patients with FH taking statins (n = 25, mean age = 57 years, 12% of males). The ALT:AST ratio was used as a marker of NAFLD risk. The mean LDL-C level was 3.38 mmol/L, and the median PCSK9 level was 312 ng/mL. The median ALT:AST ratio was 0.88. A significant negative correlation was observed between the PCSK9 and ALT:AST ratio (β = -0.67, p < 0.05). Their negative correlation might give a hypothetical insight into the effect of statin treatment on the development of NAFLD, in relation to PCSK9 behavior, in patients with FH.

Deletion of the asialyloglycoprotein receptor-1 (ASGR1) causes atheroprotective effects in vitro and in vivo

Abstract Introduction The asialoglycoprotein receptor 1 (ASGR1) is a hepatic receptor that clears desialylated glycoproteins from the circulation. A loss-of-function mutation in ASGR1 was reported to associate with a 34% reduction in coronary artery disease (CAD) risk, suggesting a role for ASGR1 in CAD. Aim To determine the role of ASGR1 in atherosclerosis and whether deletion of ASGR1 elicits atheroprotective effects. Methods Western blotting, mass spectrometry and flow cytometry assessed ASGR1 expression in cultured macrophages or immune cells collected from the blood and aortas of wildtype mice. Bone marrow-derived macrophages (BMDMs) from wildtype and Asgr1-/- mice were subjected to cholesterol efflux and oxidised low-density lipoprotein (oxLDL) uptake in vitro functionalassays. ELISA and qPCR measured changes in lipid regulators in BMDMs. Asgr1-/- mice were crossed with atherosclerosis-prone apolipoprotein (Apo)e-/- mice (Apoe-/-Asgr1-/-). Stable plaque area was assessed histologically in the aortic sinuses of mice fed a high-cholesterol diet for 8 weeks (n=16/group). Using the tandem stenosis surgical model, unstable plaque was assessed in carotid arteries (n=10/group). Human ASGR1 was measured by ELISA in peripheral blood mononuclear cells (PBMCs) isolated from patient samples (n=18). Results Western blotting and mass spectrometry discovered ASGR1 is expressed in macrophages. Immunofluorescence identified the presence of ASGR1 in aortic sinus plaque. Flow cytometry revealed ASGR1 is expressed in both inflammatory and patrolling monocytes, neutrophils, macrophages and dendritic cells of blood and aortas. Asgr1-/- BMDMs elicited greater cholesterol efflux (+39%, P&amp;lt;0.05) and decreased oxLDL uptake (-15%, P&amp;lt;0.05), compared to wildtype BMDMs. Moreover, Asgr1-/- BMDMs had significantly higher LDL receptor protein levels (+2-fold), and Lxra (+2-fold) and Pparg (+1.5-fold) mRNA levels than wildtype BMDMs, P&amp;lt;0.001. Plasma from Asgr1-/- mice had lower total (-25%, P&amp;lt;0.05) and LDL (-36%, P&amp;lt;0.05) cholesterol concentrations than wildtype mice. Apoe-/-Asgr1-/- mice developed less stable plaque in aortic sinuses (-37%, P&amp;lt;0.001) than Apoe-/- controls, as well as smaller unstable plaques in carotid arteries (-49%, P&amp;lt;0.05). Finally, in a human population, ASGR1 protein levels were higher in PBMCs from patients with coronary plaque (+61%, P&amp;lt;0.05), than those without plaque, determined from coronary angiograms. Conclusion ASGR1 is expressed on monocytes, macrophages and in plaques, and human PBMC ASGR1 associates with coronary plaque. Deletion of ASGR1 causes atheroprotective functions in macrophages in vitro and reduced plaque burden in vivo. Our findings demonstrate ASGR1 is important in atherosclerosis with implications for ASGR1 as a therapeutic target.

Engineered endothelium model enables recapitulation of vascular function and early atherosclerosis development

Human health relies heavily on the vascular endothelium. Here, we propose a novel engineered endothelium model (EEM), which recapitulated both normal vascular function and pathology. An artificial basement membrane (aBM), where porous polyvinyl alcohol hydrogel was securely integrated with human fibroblast-derived, decellularized extracellular matrix on both sides was fabricated first and followed by endothelial cells (ECs) and pericytes (PCs) adhesion, respectively. Our EEM formed robust adherens junction (VE-cad) and built an impermeable barrier with time, along with the nitric oxide (NO) secretion. In our EEM, ECs and PCs interacted each other via aBM and led to hemoglobin alpha 1 (Hb-α1) development, which was involved in NO control and was strongly interconnected with VE-cad as well. A resilient property of EEM under inflammatory milieu was also confirmed by VE-cad and barrier recovery with time. In particular interest, foam cells formation, a hallmark of atherosclerotic initiation was successfully recapitulated in our EEM, where a series of sequential events were confirmed: human monocytes adhesion, transendothelial migration, and oxidized low-density lipoprotein uptake by macrophages. Collectively, our EEM is excellent in recapitulating not only normal endothelium but early pathologic one, thereby enabling EEM to be a physiologically relevant model for vascular study and disease modeling.

ASGR1 Deficiency Inhibits Atherosclerosis in Western Diet-Fed ApoE-/- Mice by Regulating Lipoprotein Metabolism and Promoting Cholesterol Efflux.

Atherosclerosis is the most common cause of cardiovascular diseases. Clinical studies indicate that loss-of-function ASGR1 (asialoglycoprotein receptor 1) is significantly associated with lower plasma cholesterol levels and reduces cardiovascular disease risk. However, the effect of ASGR1 on atherosclerosis remains incompletely understood; whether inhibition of ASGR1 causes liver injury remains controversial. Here, we comprehensively investigated the effects and the underlying molecular mechanisms of ASGR1 deficiency and overexpression on atherosclerosis and liver injury in mice. We engineered Asgr1 knockout mice (Asgr1-/-), Asgr1 and ApoE double-knockout mice (Asgr1-/-ApoE-/-), and ASGR1-overexpressing mice on an ApoE-/- background and then fed them different diets to assess the role of ASGR1 in atherosclerosis and liver injury. After being fed a Western diet for 12 weeks, Asgr1-/-ApoE-/- mice exhibited significantly decreased atherosclerotic lesion areas in the aorta and aortic root sections, reduced plasma VLDL (very-low-density lipoprotein) cholesterol and LDL (low-density lipoprotein) cholesterol levels, decreased VLDL production, and increased fecal cholesterol contents. Conversely, ASGR1 overexpression in ApoE-/- mice increased atherosclerotic lesions in the aorta and aortic root sections, augmented plasma VLDL cholesterol and LDL cholesterol levels and VLDL production, and decreased fecal cholesterol contents. Mechanistically, ASGR1 deficiency reduced VLDL production by inhibiting the expression of MTTP (microsomal triglyceride transfer protein) and ANGPTL3 (angiopoietin-like protein 3)/ANGPTL8 (angiopoietin-like protein 8) but increasing LPL (lipoprotein lipase) activity, increased LDL uptake by increasing LDLR (LDL receptor) expression, and promoted cholesterol efflux through increasing expression of LXRα (liver X receptor-α), ABCA1 (ATP-binding cassette subfamily A member 1), ABCG5 (ATP-binding cassette subfamily G member 5), and CYP7A1 (cytochrome P450 family 7 subfamily A member 1). These underlying alterations were confirmed in ASGR1-overexpressing ApoE-/- mice. In addition, ASGR1 deficiency exacerbated liver injury in Western diet-induced Asgr1-/-ApoE-/- mice and high-fat diet-induced but not normal laboratory diet-induced and high-fat and high-cholesterol diet-induced Asgr1-/- mice, while its overexpression mitigated liver injury in Western diet-induced ASGR1-overexpressing ApoE-/- mice. Inhibition of ASGR1 inhibits atherosclerosis in Western diet-fed ApoE-/- mice, suggesting that inhibiting ASGR1 may serve as a novel therapeutic strategy to treat atherosclerosis and cardiovascular diseases.

Selective regulation of macrophage lipid metabolism via nanomaterials’ surface chemistry

Understanding the interface between nanomaterials and lipoproteins is crucial for gaining insights into their impact on lipoprotein structure and lipid metabolism. Here, we use graphene oxide (GOs) nanosheets as a controlled carbon nanomaterial model to study how surface properties influence lipoprotein corona formation and show that GOs have strong binding affinity with low-density lipoprotein (LDL). We use advanced techniques including X-ray reflectivity, circular dichroism, and molecular simulations to explore the interfacial interactions between GOs and LDL. Specifically, hydrophobic GOs preferentially associate with LDL’s lipid components, whereas hydrophilic GOs tend to bind with apolipoproteins. Furthermore, these GOs distinctly modulate a variety of lipid metabolism pathways, including LDL recognition, uptake, hydrolysis, efflux, and lipid droplet formation. This study underscores the importance of structure analysis at the nano-biomolecule interface, emphasizing how nanomaterials’ surface properties critically influence cellular lipid metabolism. These insights will inspire the design and application of future biocompatible nanomaterials and nanomedicines.

Ferritin-based disruptor nanoparticles: A novel strategy to enhance LDL cholesterol clearance via multivalent inhibition of PCSK9-LDL receptor interaction.

Hypercholesterolemia, characterized by elevated low-density lipoprotein (LDL) cholesterol levels, is a significant risk factor for cardiovascular disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in cholesterol metabolism by regulating LDL receptor degradation, making it a therapeutic target for mitigating hypercholesterolemia-associated risks. In this context, we aimed to engineer human H ferritin as a scaffold to present 24 copies of a PCSK9-targeting domain. The rationale behind this protein nanoparticle design was to disrupt the PCSK9-LDL receptor interaction, thereby attenuating the PCSK9-mediated impairment of LDL cholesterol clearance. The N-terminal sequence of human H ferritin was engineered to incorporate a 13-amino acid linear peptide (Pep2-8), which was previously identified as the smallest PCSK9 inhibitor. Exploiting the quaternary structure of ferritin, engineered nanoparticles were designed to display 24 copies of the targeting peptide on their surface, enabling a multivalent binding effect. Extensive biochemical characterization confirmed precise control over nanoparticle size and morphology, alongside robust PCSK9-binding affinity (KD in the high picomolar range). Subsequent efficacy assessments employing the HepG2 liver cell line demonstrated the ability of engineered ferritin's ability to disrupt PCSK9-LDL receptor interaction, thereby promoting LDL receptor recycling on cell surfaces and consequently enhancing LDL uptake. Our findings highlight the potential of ferritin-based platforms as versatile tools for targeting PCSK9 in the management of hypercholesterolemia. This study not only contributes to the advancement of ferritin-based therapeutics but also offers valuable insights into novel strategies for treating cardiovascular diseases.

Thrombopoietin improves the functions of bone marrow endothelial progenitor cells via METTL16/Akt signalling of haematological patients with chemotherapy-induced thrombocytopenia.

Bone marrow endothelial progenitor cells (BM EPCs) are crucial in supporting haematopoietic regeneration, while the BM EPCs of haematological patients with chemotherapy-induced thrombocytopenia (CIT) are unavoidably damaged. Therefore, the present study aimed to examine the effect of thrombopoietin (TPO) on the recovery of BM EPCs of CIT patients and to identify the underlying mechanisms. The cell functions were determined by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil)-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake and fluorescein isothiocyanate (FITC)-labeled Ulex europaeus agglutinin-I (FITC-UEA-I) binding assay, as well as proliferation, migration and tube formation experiments. Endothelial cells were transfected with METTL16 lentivirus, followed by methylated RNA immunoprecipitation sequencing. Zebrafish with vascular defect was used as the invivo model. TPO significantly improved the quantity and functions of BM EPCs from CIT patients invitro and restored the subintestinal vein area of zebrafish with vascular defect invivo. Mechanically, TPO enhanced the BM EPC functions through Akt signal mediated by METTL16, which was downregulated in BM EPCs of CIT patients and involved in the regulation of endothelial functions. The present study demonstrates that TPO improves the recovery of BM EPCs from CIT patients with haematological malignancies via METTL16/Akt signalling, which provides new insights into the role of TPO in treating CIT in addition to direct megakaryopoiesis.

Chemical constituents from the roots of Cynanchum wilfordii with PCSK9 secretion inhibitory activities

From the Cynanchum wilfordii roots, 32 compounds, including 5 previously undescribed (1, 4–6, 12) and 27 known (2, 3, 7–11, 13–32) compounds, were isolated, and their structures were elucidated using NMR spectroscopic data and MS data aided by ECD calculations or the modified Mosher's reaction. All isolates were tested for their inhibitory effects on proprotein convertase subtilisin/kexin type 9 (PCSK9) secretion. Among the isolates, compound 4, a methyl cholesterol analog, exhibited the most potent effect in reducing PCSK9 secretion, along with PCSK9 downregulation at the mRNA and protein levels via FOXO1/3 upregulation. Moreover, compound 4 attenuated statin-induced PCSK9 expression and enhanced the uptake of DiI-LDL low-density lipoprotein. Thus, compound 4 is suggested to be a potential candidate for controlling cholesterol levels.

20( S )-Protopanaxatriol Improves Atherosclerosis by Inhibiting Low-Density Lipoprotein Receptor Degradation in ApoE KO Mice.

Atherosclerosis (AS) is a chronic progressive disease caused by various factors and causes various cerebrovascular and cardiovascular diseases (CVDs). Reducing the plasma levels of low-density lipoprotein cholesterol is the primary goal in preventing and treating AS. Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a crucial role in regulating low-density lipoprotein cholesterol metabolism. Panax notoginseng has potent lipid-reducing effects and protects against CVDs, and its saponins induce vascular dilatation, inhibit thrombus formation, and are used in treating CVDs. However, the anti-AS effect of the secondary metabolite, 20( S )-protopanaxatriol (20( S )-PPT), remains unclear. In this study, the anti-AS effect and molecular mechanism of 20( S )-PPT were investigated in vivo and in vitro by Western blotting, real-time polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence staining, and other assays. The in vitro experiments revealed that 20( S )-PPT reduced the levels of PCSK9 in the supernatant of HepG2 cells, upregulated low-density lipoprotein receptor protein levels, promoted low-density lipoprotein uptake by HepG2 cells, and reduced PCSK9 mRNA transcription by upregulating the levels of forkhead box O3 protein and mRNA and decreasing the levels of HNF1α and SREBP2 protein and mRNA. The in vivo experiments revealed that 20( S )-PPT upregulated aortic α-smooth muscle actin expression, increased the stability of atherosclerotic plaques, and reduced aortic plaque formation induced by a high-cholesterol diet in ApoE -/- mice (high-cholesterol diet-fed group). Additionally, 20( S )-PPT reduced the aortic expression of CD68, reduced inflammation in the aortic root, and alleviated the hepatic lesions in the high-cholesterol diet-fed group. The study revealed that 20( S )-PPT inhibited low-density lipoprotein receptor degradation via PCSK9 to alleviate AS.

Scavenger Receptors: Different Classes and their Role in the Uptake of Oxidized Low-Density Lipoproteins

Oxidation of lipoproteins marks a fundamental early phase in atherosclerosis development, a condition distinguished by plaque build-up in the arteries. It is widely accepted that the oxidation of low-density lipoprotein (LDL) plays a pivotal role in atherosclerosis progression. Oxidized LDL (Ox-LDL) exhibits numerous atherogenic characteristics, leading to endothelial dysfunction, the creation of foam cells, and inflammation in the arterial wall. The crucial interaction between Ox-LDL and specific receptors on endothelial cells is key to initiating these processes. In this article, we're going to delve into the various LDL receptors that play a crucial part in the uptake and metabolism of LDL, emphasizing their significance in the development of atherosclerosis. The Cochrane database, Embase, PubMed, Scopus, Google Scholar, Ovid, and other databases were thoroughly searched for works addressing scavenger receptors to explore how both native LDL (nLDL) and Ox-LDL engage with these receptors, facilitating the formation and progression of atherosclerotic plaques. We'll then proceed to a more detailed examination of the complex role scavenger receptors play in the uptake and internalization of oxidized low-density lipoproteins (Ox-LDL), and their vital role in the onset of diseases. The review will further cover the identification, categorization, and roles of scavenger receptors, along with their molecular mechanisms. Grasping the complexity of these receptor-ligand interactions is critical for the creation of focused treatments to fight against pathologic conditions such as atherosclerosis and its related health scenarios.

Investigating layer-by-layer chitosan-dextran sulfate-coated mesoporous silica as a pH-sensitive drug delivery system

Mesoporous silica nanoparticles (MSNPs) coated by chitosan (CS) were shown to be a proper candidate as a carrier for drug delivery purposes. However, choosing the suitable drug-containing complexes to be applied on MSNPs-CS is of much greater importance to evaluate the possible candidate for an efficient combination of cell viability, drug release kinetics, and atherosclerosis prevention. In this regard, this study concentrates on the synthesis and assessment of coated MSNPs-CS designed for drug delivery purposes. The MSNPs are coated with polyelectrolyte complexes (PEC) composed of CS and dextran sulfate (MSNPs-CS-DX), serving as a versatile drug carrier with favorable biological characteristics. CS-DX is applied to MSNPs without requiring complex or multi-step synthesis procedures. Rosuvastatin, a cholesterol-lowering medication, is chosen for its therapeutic relevance. Additionally, CS-DX is found to relatively impede the uptake of low-density lipoproteins (LDLs) by macrophages, enhancing their potential therapeutic utility. FTIR pattern, FESEM, and TEM images prove MSNPs-CS-DX formation. DLS measurement demonstrates the average particle size of 110 nm for MSNPs, with the combined thickness of CS and DX layers ranging from 10 to 15 nm. BET test is carried out to evaluate the pore size and porosity of structure, showing outstanding results that cause an entrapment efficiency of 57% for MSNPs-CS-DX. Furthermore, the findings demonstrate the pH sensitivity of MSNPs-CS-DX on drug release kinetics. Notably, the CS-DX layer exhibits a significant enhancement in cell viability of human umbilical vein endothelial cells (HUVEC) by approximately 24% within a 24 h timeframe compared to MSNPs lacking CS-DX.Graphical

PGC-1α repression dysregulates lipid metabolism and induces lipid droplet accumulation in the retinal pigment epithelium

Drusen, the yellow deposits under the retina, are composed of lipids and proteins, and represent a hallmark of age-related macular degeneration (AMD). Lipid droplets are also reported in the retinal pigment epithelium (RPE) from AMD donor eyes. However, the mechanisms underlying these disease phenotypes remain elusive. Previously, we showed that Pgc-1α repression, combined with a high-fat diet (HFD), induce drastic AMD-like phenotypes in mice. We also reported increased PGC-1α acetylation and subsequent deactivation in the RPE derived from AMD donor eyes. Here, through a series of in vivo and in vitro experiments, we sought to investigate the molecular mechanisms by which PGC-1α repression could influence RPE and retinal function. We show that PGC-1α plays an important role in RPE and retinal lipid metabolism and function. In mice, repression of Pgc-1α alone induced RPE and retinal degeneration and drusen-like deposits. In vitro inhibition of PGC1A by CRISPR-Cas9 gene editing in human RPE (ARPE19- PGC1A KO) affected the expression of genes responsible for lipid metabolism, fatty acid β-oxidation (FAO), fatty acid transport, low-density lipoprotein (LDL) uptake, cholesterol esterification, cholesterol biosynthesis, and cholesterol efflux. Moreover, inhibition of PGC1A in RPE cells caused lipid droplet accumulation and lipid peroxidation. ARPE19-PGC1A KO cells also showed reduced mitochondrial biosynthesis, impaired mitochondrial dynamics and activity, reduced antioxidant enzymes, decreased mitochondrial membrane potential, loss of cardiolipin, and increased susceptibility to oxidative stress. Our data demonstrate the crucial role of PGC-1α in regulating lipid metabolism. They provide new insights into the mechanisms involved in lipid and drusen accumulation in the RPE and retina during aging and AMD, which may pave the way for developing novel therapeutic strategies targeting PGC-1α.

Epigallocatechin gallate induces an up-regulation of LDLR accompanied by a reduction of idol in Hepg2 cells

AbstractEpigallocatechin gallate (EGCG), the primary catechin in green tea, has improved cholesterol metabolism. However, the molecular mechanisms of EGCG underlying these functions are not fully understood. In this study, we aimed to investigate the molecular mechanisms underlying EGCG’s effect on low-density lipoprotein (LDL) in HepG2 cells. Real-time PCR and Western blot analysis were used to determine the mRNA and protein levels in the human hepatoma cell line (HepG2). LDL uptake assay was used to quantify the low-density lipoprotein receptor (LDLR) function. EGCG induced significantly up-regulated LDLR protein and mRNA levels in HepG2 cells (P &lt; 0.05). Both at the transcriptional level and at the protein level, EGCG can significantly (P &lt; 0.05) down-regulate the elevated expression levels of liver X receptor α (LXRα) and inducible degrader of the LDLR (Idol) due to 25-OHC. Fluorescence results showed that EGCG induction could also significantly increase LDL uptake (P &lt; 0.05). EGCG regulates LDL uptake through the LXRα-LDLR pathway, and EGCG can effectively improve the abnormal expression of protein and mRNA induced by 25-OHC. Graphical abstract

Trem2 Agonist Reprograms Foamy Macrophages to Promote Atherosclerotic Plaque Stability-Brief Report.

Trem2 (triggering receptor on myeloid cells 2), a surface lipid receptor, is expressed on foamy macrophages within atherosclerotic lesions and regulates cell survival, proliferation, and anti-inflammatory responses. Studies examining the role of Trem2 in atherosclerosis have shown that deletion of Trem2 leads to impaired foamy macrophage lipid uptake, proliferation, survival, and cholesterol efflux. Thus, we tested the hypothesis that administration of a Trem2 agonist antibody (AL002a) to atherogenic mice would enhance macrophage survival and decrease necrotic core formation to improve plaque stability. To model a therapeutic intervention approach, atherosclerosis-prone mice (Ldlr [low-density lipoprotein receptor]-/-) were fed a high-fat diet for 8 weeks, then transitioned to treatment with AL002a or isotype control for an additional 8 weeks while continuing on a high-fat diet. AL002a-treated mice had increased lesion size in both the aortic root and whole mount aorta, which correlated with an expansion of plaque macrophage area. This expansion was due to increased macrophage survival and proliferation in plaques. Importantly, plaques from AL002a-treated mice showed improved features of plaque stability, including smaller necrotic cores, increased fibrous caps, and greater collagen deposition. Single-cell RNA sequencing of whole aorta suspensions from isotype- and AL002a-treated atherosclerotic mice revealed that Trem2 agonism dramatically altered foamy macrophage transcriptome. This included upregulation of oxidative phosphorylation and increased expression of collagen genes. In vitro studies validated that Trem2 agonism with AL002a promoted foamy macrophage oxidized low-density lipoprotein uptake, survival, and cholesterol efflux. Trem2 agonism expands atherosclerotic plaque macrophages by promoting cell survival and proliferation but improves features of plaque stability by rewiring foamy macrophage function to enhance cholesterol efflux and collagen deposition.

Abstract 2046: Overexpression Of Endothelial Lipase Enhances The Cellular Uptake Of Low-density Lipoproteins In Cultured Hepatocytes In The Absence Of Low-density Lipoprotein Receptor.

Background: Inhibitors of angiopoietin-like3 (ANGPTL3) reduce low-density lipoprotein (LDL) cholesterol, even in Familial Hypercholesterolemia (FH) patients with a dysfunctional LDL receptor (LDLR). Although the exact mechanism remains elusive, it may depend upon endothelial lipase (EL). Early studies showed that EL mediates the cellular uptake of high-density lipoproteins in hepatocytes; however, whether EL similarly mediates LDL uptake is unknown. Aim: Investigate the role of EL in mediating the cellular uptake of LDL in an LDLR-deficient model. Methods: Cas9-expressing HepG2 cells were transfected with a scrambled or an LDLR -targeting guide RNA to produce control and LDLR -knockout (KO) cells. Control and KO cells were transfected by lipofection with an empty plasmid or a plasmid containing the human LIPG gene (EL). Cellular uptake of fluorescent human LDL was measured by FACS. In addition, we measured the uptake of LDL in cells with and without the pre-incubation with 5 U/ml heparin or a cocktail of heparinases I, II, and II to test the contribution of heparan sulfate proteoglycans (HSPG). Results: LDLR -KO HepG2 cells showed a 20-25% residual uptake of LDL compared to controls (p&lt;0.001) and 10-fold higher levels of EL mRNA. Remarkably, LIPG -transfected LDLR -KO hepatocytes showed a 2-fold increase in LDL uptake compared to LDLR-KO cells non-overexpressing EL (p&lt;0.001). The pre-incubation with either heparinases or heparin completely blocked the uptake of LDL not only in LIPG -transfected LDLR -KO cells but also in those LDLR -KO cells transfected with the empty plasmid, suggesting a crucial role of HSPG in the LDL uptake. Conversely, overexpression of EL decreased LDL uptake in control cells (-11%, p=0.02), while unchanged or slightly increased LDL uptake was observed after incubating with heparinases or heparin. Conclusions: Our findings demonstrate that EL facilitates the uptake of LDL through an LDLR-independent, HSPG-dependent pathway. This pathway may be responsible for the residual uptake of LDL observed in LDLR -KO hepatocytes and may represent a potential druggable target to treat FH. Our data provides an alternative mechanism to explain the reduction of LDL cholesterol induced by ANGPTL3 inhibitors.

A Sexually Dimorphic Hepatic Cycle of Very Low Density Lipoprotein Uptake and Assembly

The liver shows striking sexual dimorphism, which is reflected in differences between women and men in pathologies inherently associated with hepatic function. Recent single-cell transcriptomes revealed spatiotemporal programmes of liver function on the sublobular scale. However, how sexual dimorphism affects this space-time logic remains poorly understood. To address this, we performed single-cell RNA-seq in the mouse liver, which allowed us to model how lobular position, circadian time and sex shape the transcriptome. We found that sex, space and time markedly influence xenobiotic detoxification and lipid processing, including lipoprotein metabolism. Crucially, the very low density lipoprotein receptor (VLDLR) is restricted to the pericentral zone, with significantly higher mRNA and protein levels in female mice. Using human samples, we were able to recapitulate both VLDLR's pericentral expression as well as higher mRNA and protein levels in premenopausal women compared to similarly aged men. In humans, we found that VLDLR expression depends not just on age but also body mass index. Conversely, several genes involved in VLDL assembly are periportally biased, and we corroborated periportal VLDL generation with electron microscopy, optimized for detecting low density lipid particles. Together, this suggests a hepatic cycle of periportal formation and pericentral uptake of VLDL. Crucially, these processes provide a new window into the sexual dimorphism characterizing differences in VLDL kinetics and atherosclerotic cardiovascular disease. The work was supported by a Swiss National Science Foundation grant. The authors declare no conflicting interests. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 115: Scavenger Receptor-BI Mediates Endothelial Uptake Of Lipoprotein(a)

Elevated levels of lipoprotein(a) [Lp(a)], an apoB100-containing lipoprotein, are an independent risk factor for atherosclerotic cardiovascular disease, even in the setting of effective reduction of plasma low-density lipoprotein (LDL) cholesterol. To date, the mechanisms whereby Lp(a) exerts its atherogenic effects remain poorly understood, and no treatments aimed at lowering Lp(a) have been approved. Two receptors - scavenger receptor-BI (SR-BI) and activin receptor-like kinase 1 (ALK1) - have been proposed to bind apoB100 and mediate the atherogenic uptake and transcytosis of LDL across the endothelial cell (EC) barrier. EC-specific SR-BI knockout reduces atherosclerosis in both apoE and LDLR knockout mice. We have shown that SR-BI also mediates EC uptake of nascent chylomicrons. Although binding of ALK1 to apoB is believed to occur through the N-terminus of apoB, the receptor is not involved in the uptake of chylomicrons. We assessed which if any of these receptors are involved in Lp(a) uptake by ECs. Despite the presence of apoB100, EC uptake of Lp(a), like apoB48 chylomicrons, was reduced by SR-BI, but not ALK1, knockdown. We then used a series of small N terminal apoB peptides (apoB3-apoB18, with sequences comprising the 3%, 8%, 12%, 15%, and 18% N terminus of apoB) to compete with Lp(a) uptake. Both chylomicron and Lp(a) uptake was inhibited by apoB15 and apoB18, whereas LDL uptake was additionally inhibited by apoB12. These observations suggest that a) the binding sequence to SR-BI is between residues 547-684 of apoB, and b) an additional sequence between residues 365-547 participates in apoB binding to ALK1. Our findings suggest that apo(a) alters the structure of apoB to make it a better ligand for SR-BI uptake by ECs. Because this uptake pathway leads to transcytosis, we hypothesize that the greater atherogenicity of Lp(a) is mediated by its increased binding to and uptake by ECs.