High-density Lipoprotein Receptor Research Articles

Nanoparticle-Directed Antioxidant Therapy Can Ameliorate Disease Progression in a Novel, Diet-Inducible Model of Coronary Artery Disease.

Oxidative stress plays a crucial role in the pathogenesis of coronary artery disease. In cardiovascular research using murine models, the generation and maintenance of models with robust coronary arterial atherosclerosis has been challenging. We characterized a new mouse model in which the last 3 amino acids of the carboxyl terminus of the HDL (high-density lipoprotein) receptor (SR-B1 [scavenger receptor class B, member 1]) were deleted in a low-density lipoprotein receptor knockout (LDLR-/-) mouse model (SR-B1ΔCT/LDLR-/-) fed an atherogenic diet. We also tested the therapeutic effects of an oxidative stress-targeted nanoparticle in atherogenic diet-fed SR-B1ΔCT/LDLR-/- mice. The SR-B1ΔCT/LDLR-/- mice fed an atherogenic diet had occlusive coronary artery atherosclerosis, impaired cardiac function, and a dramatically lower survival rate, compared with LDLR-/- mice fed the same diet. As SR-B1ΔCT/LDLR-/- mice do not exhibit female infertility or low pup yield, they are far easier and less costly to use than the previously described SR-B1-based models of coronary artery disease. We found that treatment with the targeted nanoparticles improved the cardiac functions and corrected hematologic abnormalities caused by the atherogenic diet in SR-B1ΔCT/LDLR-/- mice but did not alter the distinctive plasma lipid levels. The SR-B1ΔCT/LDLR-/- mice developed diet-inducible, fatal atherosclerotic coronary artery disease, which could be ameliorated by targeted nanoparticle therapy. Our study provides new tools for the development of cardiovascular therapies.

Sirtuin 1-mediated autophagy regulates testosterone synthesis in Leydig cells of piglets

Testosterone is secreted by Leydig cells (LCs), which play an important physiological role in preserving male secondary sex characteristics, protecting male reproductive function, and establishing the blood-testis barrier. Studies have shown that autophagy is particularly active in LCs; however, its involvement in testosterone synthesis in porcine LCs has not been fully explored. Therefore, this experiment aimed to investigate the influence of autophagy on testosterone secretion in porcine LCs and its potential regulatory mechanism. Our results demonstrated that both testicular autophagy and serum testosterone levels increased in piglets during postnatal development from 4 to 18 weeks. In addition, autophagy was found to degrade the Na+/H+ exchange regulatory factor 2 (NHERF2), leading to the up-regulation of scavenger receptor class B type 1 (SRB1). This process resulted in increased cholesterol intake and enhanced testosterone production. The observable level of sirtuin 1 (SIRT1) was directly proportional to the level of autophagy. In vitro investigations have shown that SIRT1 can affect the level of autophagy, cholesterol uptake as well as testosterone release. In conclusion, testosterone synthesis during pig development is regulated by SIRT1. SIRT1 mediates the degradation of NHERF2 through autophagy, thereby weakening its negative regulatory effect on the high-density lipoprotein receptor SRB1 in Leydig cells. This process increases cholesterol uptake and enhances testosterone synthesis.

Protein Corona-Directed Cellular Recognition and Uptake of Polyethylene Nanoplastics by Macrophages.

The widespread use of plastic products in daily life has raised concerns about the health hazards associated with nanoplastics (NPs). When exposed, NPs are likely to infiltrate the bloodstream, interact with plasma proteins, and trigger macrophage recognition and clearance. In this study, we focused on establishing a correlation between the unique protein coronal signatures of high-density (HDPE) and low-density (LDPE) polyethylene (PE) NPs with their ultimate impact on macrophage recognition and cytotoxicity. We observed that low-density and high-density lipoprotein receptors (LDLR and SR-B1), facilitated by apolipoproteins, played an essential role in PE-NP recognition. Consequently, PE-NPs activated the caspase-3/GSDME pathway and ultimately led to pyroptosis. Advanced imaging techniques, including label-free scattered light confocal imaging and cryo-soft X-ray transmission microscopy with 3D-tomographic reconstruction (nano-CT), provided powerful insights into visualizing NPs-cell interactions. These findings underscore the potential risks of NPs to macrophages and introduce analytical methods for studying the behavior of NPs in biological systems.

Ligand-based Molecular Modeling of HDL Receptor SR-BI Inhibitors As Potent Anti-Hyperlipidemic Agents

Introduction:: The High-density lipoprotein (HDL) receptor, Scavenger receptor class B, type I (SRBI) plays a crucial role in lipoprotein metabolism, cholesterol homeostasis, and atherosclerosis. In the present study, a quantitative structure-activity relationship study (QSAR) investigation was conducted on a data set of 31 novel indolinyl thiazole-based inhibitors of SR-BI mediated lipid uptake. Method:: To build the QSAR model, Multiple linear regression analysis (MLR), partial least square analysis (PLS), and neural analysis (NN) were performed which were further evaluated internally as well as externally for the prediction of activity. The best QSAR model for MLR was selected with a correlation coefficient (r2) of 0.937, cross-validation r2cv of 0.908, and a standard error (S) value of 0.253. For PLS, r2 was 0.937 and for FFNN r2 was 0.961 (for the training set). This was further evaluated externally by a test set having r2 values 0.870 (MLR), 0.863(PLS), and 0.933(neural network) analysis. Result:: The final model comprised hydrophobic parameters (Lipole Z component) and steric parameters (molar refractivity and K alpha2 index). Conclusion:: All these descriptors generated comparable results which prove that the model generated is sound and has good predictability.

Effect of Gracilaria changii extract and its fraction in reverse cholesterol transport by targeting SR-BI gene

This present study aimed to investigate the effects of Gracilaria changii (Red seaweed) extract and its fraction on scavenger receptor class B type I (SR-BI) gene expressions. In vitro studies of the activity of G. changii extract and its fraction against the therapeutic target for hypocholesterolemia were conducted using high-density lipoprotein (HDL) receptor SR-BI via luciferase assay in HepG2 cells. In the current study, methanol crude extract (MCE) and Fraction-2 of G. changii showed the highest expression levels of the SR-BI gene via luciferase assay and increased SR-BI mRNA expression compared to the negative control. Metabolite profiling of the MCE of G. changii by GC-MS revealed nine major compounds, comprising various fatty acids, particularly hexadecenoic acid. Therefore, the MCE and Fraction-2 of G. changii have the potential to be explored in the treatment of hypercholesterolaemia to prevent or reduce the severity of atherosclerosis development.

129 Selenium-form affects de novo cholesterol synthesis and catabolism of cholesterol and progesterone in the liver of beef heifers

Abstract A deficiency of selenium (Se) in soils and hence forages poses a challenge to producers in the southeast United States, necessitating supplementation of this trace mineral to the diet of pasture fed cattle. Se occurs in either inorganic (ISe) or organic (OSe) forms; ISe is conventionally available in commercial supplements, while OSe is available in grazed forages. We have investigated supplementation with either ISe or a 1:1 mixture of ISe:OSe (MIX) and demonstrated that MIX increases the relative abundance of mRNA encoding the low-density lipoprotein receptor (LDLR) and enzymes involved in de novo cholesterol synthesis in the corpus luteum, concurrent with a MIX-induced increase in systemic progesterone (P4) during the early luteal phase. We also observed a MIX-induced decrease in circulating cholesterol and low-density/very low-density lipoproteins during early gestation, and a MIX-induced increase in conceptus length at maternal recognition of pregnancy (MRP). The objective herein was to investigate the effect of form of Se (ISe versus MIX) on de novo cholesterol synthesis and cholesterol/steroid catabolism in the liver at MRP. We hypothesized form of Se would affect the abundance of transcripts that regulate synthesis, catabolism, and local concentrations of cholesterol and P4. Angus-cross heifers were supplemented with ISe or MIX for at least 90 d before insemination at observed estrus. On d 17 of pregnancy, heifers (n = 6/TRT) were killed and their reproductive tracts and conceptuses recovered. Hepatic samples were collected for qPCR analysis of mRNA transcripts encoding proteins involved in de novo cholesterol synthesis, regulation of cholesterol availability and steroid catabolism. Additionally, on d 7 and d 17 of pregnancy, we quantified systemic concentrations of high-density lipoproteins (HDL), indicative of cholesterol recycling to the liver. We quantified the relative abundance of 22 mRNAs and observed that heifers supplemented with MIX-form Se had an increase in the relative abundance of mRNA encoding the very low-density lipoprotein receptor (VLDLR, P = 0.01). Unexpectedly, we failed to detect an effect of TRT on the abundance of mRNA encoding the high-density lipoprotein receptor (SCARB1, P > 0.05) which was consistent with our inability to detect an effect on the serum concentration of total or free HDL (P > 0.05) during early pregnancy and at MRP. Of the 11 mRNA transcripts encoding enzymes involved in catabolism of cholesterol and progesterone, including steroid alpha reductases (SRD5A1 and SRD5A3) and cytochrome P450 enzymes (CYP4A2, CYP4A11, CYP11A1, and CYP17A1), we were unable to detect a difference in the relative abundance of any transcript. Overall, it appears that the form of Se is altering circulating concentrations of cholesterol and further affecting whole animal physiology and fertility; however, the mechanism involved remains to be fully elucidated and warrants further investigation.

PON1 has palmitoyl-protein thioesterase (PPT) activity, and can affect the presence of SR-B1 on the endothelial cell membrane.

The high-density lipoprotein (HDL)-associated enzyme paraoxonase 1 (PON1) is expressed almost exclusively in the liver and is then transported by HDL to the peripheral tissues. The lipophilic nature of PON1 enables its easy exchange between the lipoprotein and cell membranes in a process that is dependent on the HDL receptor scavenger receptor class B, type 1 (SR-B1). In endothelial cells, PON1 binding to the cell membrane leads to its internalization by endocytosis and subsequent lysosomal degradation. PON1 is a "promiscuous" enzyme with unusually broad substrate specificity in vitro, but its actual function and substrate are still unknown. The enzyme requires a lipid environment and becomes completely inactive upon delipidation. However, when PON1 binds HDL, its active site faces the lipoprotein's core and is inaccessible to external substrates. Hence, the HDL-bound PON1 is inactive toward substrates outside the particle's lipid core and is rapidly degraded and becomes inactive upon internalization. Consequently, the enzyme is only active in the cell membrane during its transit from HDL to the cytoplasm. To assign a function to PON1, we investigated whether it is a palmitoyl-protein thioesterase (PPT) that can hydrolyze the palmitoyl moieties of membrane proteins involved in HDL and cholesterol transport, such as SR-B1, ABCA1, or their neighboring caveola proteins to facilitate the release of HDL or trigger its endocytosis. This study shows that PON1 can hydrolyze palmitoyl-cysteine thioester bonds in vitro, has direct or indirect PPT activity in vivo, and can significantly decrease the presence of SR-B1 in the endothelial membrane.

Patulin Stimulates Progenitor Leydig Cell Proliferation but Delays Its Differentiation in Male Rats during Prepuberty.

Patulin is a mycotoxin with potential reproductive toxicity. We explored the impact of patulin on Leydig cell (LC) development in male rats. Male Sprague Dawley rats (21 days postpartum) were gavaged patulin at doses of 0.5, 1, and 2 mg/kg/day for 7 days. Patulin markedly lowered serum testosterone at ≥0.5 mg/kg and progesterone at 1 and 2 mg/kg, while increasing LH levels at 2 mg/kg. Patulin increased the CYP11A1+ (cholesterol side-chain cleavage, a progenitor LC biomarker) cell number and their proliferation at 1 and 2 mg/kg. Additionally, patulin downregulated Lhcgr (luteinizing hormone receptor), Scarb1 (high-density lipoprotein receptor), and Cyp17a1 (17α-hydroxylase/17,20-lyase) at 1 and 2 mg/kg. It increased the activation of pAKT1 (protein kinase B), pERK1/2 (extracellular signal-related kinases 1 and 2), pCREB (cyclic AMP response binding protein), and CCND1 (cyclin D1), associated with cell cycle regulation, in vivo. Patulin increased EdU incorporation into R2C LC and stimulated cell cycle progression in vitro. Furthermore, patulin showed a direct inhibitory effect on 11β-HSD2 (11β-hydroxysteroid dehydrogenase 2) activity, which eliminates the adverse effects of glucocorticoids. This study provides insights into the potential mechanisms via which patulin affects progenitor LC development in young male rats.

Development and validation of a purification system for functional full-length human SR-B1 and CD36

Scavenger receptor class B type 1 (SR-B1) and CD36 are both members of the class B scavenger receptor family that play important roles in lipoprotein metabolism and atherosclerotic disease. SR-B1 is the primary receptor for high-density lipoproteins, while CD36 is the receptor responsible for the internalization of oxidized low-density lipoproteins. Despite their importance, class B scavenger receptor structure has only been studied by functional domain or peptide fragments-there are currently no reports of utilizing purified full-length protein. Here we report the successful expression and purification of full-length human SR-B1 and CD36 using an Spodoptera frugiperda insect cell system. We demonstrate that both SR-B1 and CD36 retained their normal functions in Spodoptera frugiperda cells, including lipoprotein binding, lipid transport, and the formation of higher order oligomers in the plasma membrane. Purification schemes for both scavenger receptors were optimized and their purity was confirmed by SDS-PAGE. Both purified scavenger receptors were assessed for stability by thermal shift assay and shown to maintain stable melting temperatures up to 6weeks post-purification. Microscale thermophoresis was used to demonstrate that purified SR-B1 and CD36 were able to bind their native lipoprotein ligands. Further, there was no difference in affinity of SR-B1 for high-density lipoprotein or CD36 for oxidized low-density lipoprotein, when comparing glycosylated and deglycosylated receptors. These studies mark a significant step forward in creating physiologically relevant tools to study scavenger receptor function and lay the groundwork for future functional studies and determination of receptor structure.

Deletion of the scavenger receptor Scarb1 in myeloid cells does not affect bone mass.

The scavenger receptor class B member 1 (SR-B1 or Scarb1) is a glycosylated cell surface receptor for high density lipoproteins (HDL), oxidized low density lipoproteins (OxLDL), and phosphocholine-containing oxidized phospholipids (PC-OxPLs). Scarb1 is expressed in macrophages and has been shown to have both pro- and anti-atherogenic properties. It has been reported that global deletion of Scarb1 in mice leads to either high or low bone mass and that PC-OxPLs decrease osteoblastogenesis and increase osteoclastogenesis. PC-OxPLs decrease bone mass in 6-month-old mice and are critical pathogenetic factors in the bone loss caused by high fat diet or aging. We have investigated here whether Scarb1 expression in myeloid cells affects bone mass and whether PC-OxPLs exert their anti-osteogenic effects via activation of Scarb1 in macrophages. To this end, we generated mice with deletion of Scarb1 in LysM-Cre expressing cells and found that lack of Scarb1 did not affect bone mass in vivo. These results indicate that Scarb1 expression in cells of the myeloid/osteoclast lineage does not contribute to bone homeostasis. Based on this evidence, and earlier studies of ours showing that Scarb1 expression in osteoblasts does not affect bone mass, we conclude that Scarb1 is not an important mediator of the adverse effects on PC-OxPLs in osteoclasts or osteoblasts in 6-month-old mice.

The potential role of scavenger receptor B type I (SR-BI) in SARS-CoV-2 infection.

Scavenger receptor type B I (SR-BI), the major receptor for high-density lipoprotein (HDL) mediates the delivery of cholesterol ester and cholesterol from HDL to the cell membrane. SR-BI is implicated as a receptor for entry of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). SR-BI is colocalized with the angiotensin-converting enzyme 2 (ACE2) increasing the binding and affinity of SARS-CoV-2 to ACE2 with subsequent viral internalization. SR-BI regulates lymphocyte proliferation and the release of pro-inflammatory cytokines from activated macrophages and lymphocytes. SR-BI is reduced during COVID-19 due to consumption by SARS-CoV-2 infection. COVID-19-associated inflammatory changes and high angiotensin II (AngII) might be possible causes of repression of SR-BI in SARS-CoV-2 infection. In conclusion, the downregulation of SR-BI in COVID-19 could be due to direct invasion by SARS-CoV-2 or through upregulation of pro-inflammatory cytokines, inflammatory signaling pathways, and high circulating AngII. Reduction of SR-BI in COVID-19 look like ACE2 may provoke COVID-19 severity through exaggeration of the immune response. Further studies are invoked to clarify the potential role of SR-BI in the pathogenesis of COVID-19 that could be protective rather than detrimental.

The adrenal stress response is an essential host response against therapy-induced lethal immune activation.

Cytokine release syndrome (CRS) is a systemic inflammatory syndrome associated with infection- or drug-induced T cell activation and can cause multiple organ failure and even death. Because current treatments are ineffective in some patients with severe CRS, we set out to identify risk factors and mechanisms behind severe CRS that might lead to preventive therapies and better clinical outcomes in patients. In mice, we found that deficiency in the adrenal stress response-with similarities to such in patients called relative adrenal insufficiency (RAI)-conferred a high risk for lethal CRS. Mice treated with CD3 antibodies were protected against lethal CRS by the production of glucocorticoids (GC) induced by the adrenal stress response in a manner dependent on the scavenger receptor B1 (SR-BI), a receptor for high-density lipoprotein (HDL). Mice with whole-body or adrenal gland-specific SR-BI deficiency exhibited impaired GC production, more severe CRS, and increased mortality in response to CD3 antibodies. Pretreatment with a low dose of GC effectively suppressed the development of CRS and rescued survival in SR-BI-deficient mice without compromising T cell function through apoptosis. Our findings suggest that RAI may be a risk factor for therapy-induced CRS and that pretreating RAI patients with GC may prevent lethal CRS.

Zebrafish as outgroup model to study evolution of scavenger receptor class B type I functions

Background and aimsScavenger receptor class B1 (SCARB1) - also known as the high-density lipoprotein (HDL) receptor - is a multi-ligand scavenger receptor that is primarily expressed in liver and steroidogenic organs. This receptor is known for its function in reverse cholesterol transport (RCT) in mammals and hence disruption leads to a massive increase in HDL cholesterol in these species. The extracellular domain of SCARB1 - which is important for cholesterol handling - is highly conserved across multiple vertebrates, except in zebrafish. Methods: To examine the functional conservation of SCARB1 among vertebrates, two stable scarb1 knockout zebrafish lines, scarb1 715delA (scarb1 −1 nt) and scarb1 715_716insGG (scarb1 +2 nt), were created using CRISPR-Cas9 technology. ResultsWe demonstrate that, in zebrafish, SCARB1 deficiency leads to disruption of carotenoid-based pigmentation, reduced fertility, and a decreased larvae survival rate, whereas steroidogenesis was unaltered. The observed reduced fertility is driven by defects in female fertility (−50 %, p < 0.001). Importantly, these alterations were independent of changes in free (wild-type 2.4 ± 0.2 μg/μl versus scarb1−/− 2.0 ± 0.1 μg/μl) as well as total (wild-type 4.2 ± 0.4 μg/μl versus scarb1−/− 4.0 ± 0.3 μg/μl) plasma cholesterol levels. Uptake of HDL in the liver of scarb1−/− zebrafish larvae was reduced (−86.7 %, p < 0.001), but this coincided with reduced perfusion of the liver. No effect was observed on lipoprotein uptake in the caudal vein. SCARB1 deficient canaries, which also lack carotenoids in their plumage, similarly as scarb1−/− zebrafish, failed to show an increase in plasma free- and total cholesterol levels. ConclusionOur findings suggest that the specific function of SCARB1 in maintaining plasma cholesterol could be an evolutionary novelty that became prominent in mammals, while other known functions were already present earlier during vertebrate evolution.

Extensive diet-induced atherosclerosis in scavenger receptor class B type 1-deficient mice is associated with substantial leukocytosis and elevated vascular cell adhesion molecule-1 expression in coronary artery endothelium.

High levels of low density lipoprotein (LDL) cholesterol and low levels of high density lipoprotein (HDL) cholesterol are risk factors for cardiovascular disease. Mice that lack genes involved in the clearance of LDL from the bloodstream, such as the LDL receptor and apolipoprotein E, are widely used models of experimental atherosclerosis. Conversely, mice that lack the HDL receptor, scavenger receptor class B type I, and therefore have disrupted HDL functionality, also develop diet-inducible atherosclerosis but are a seldom-used disease model. In this study, we compared atherosclerosis and associated phenotypes in scavenger receptor class B type I knockout mice with those of wild type, LDL receptor knockout, and apolipoprotein E knockout mice after 20weeks of being fed an atherogenic diet containing sodium cholate. We found that while scavenger receptor class B type I knockout mice had substantially lower plasma cholesterol than LDL receptor and apolipoprotein E knockout mice, they developed atherosclerotic plaques with similar sizes and compositions in their aortic sinuses, and more extensive atherosclerosis in their descending aortas and coronary arteries. This was associated with elevated tumor necrosis factor alpha levels in scavenger receptor class B type I knockout mice compared to wild type and LDL receptor knockout mice, and lymphocytosis, monocytosis, and elevated vascular cell adhesion molecule expression in coronary artery endothelial cells compared to the other mice examined. We conclude that extensive atherosclerosis in arteries that are not generally susceptible to atherosclerosis in scavenger receptor class B type I knockout mice is driven by factors in addition to hypercholesterolemia, including inflammation, dysregulation of the immune system and increased sensitivity of endothelial cells in arteries that are normally resistant to atherosclerosis. Scavenger receptor class B type I knockout mice fed a cholate containing atherogenic diet may prove to be a useful model to study mechanisms of atherosclerosis and evaluate treatments that rely on intact LDL clearance pathways.

Targeting Ligand Independent Tropism of siRNA-LNP by Small Molecules for Directed Therapy of Liver or Myeloid Immune Cells.

Hepatic clearance of lipid nanoparticles (LNP) with encapsulated nucleic acids restricts their therapeutic applicability. Therefore, tools for regulating hepatic clearance are of high interest for nucleic acid delivery. To this end, this work employs wild-type (WT) and low-density lipoprotein receptor (Ldlr)-/- mice pretreated with either a leukotriene B4 receptor inhibitor (BLT1i) or a high-density lipoprotein receptor inhibitor (HDLRi) prior to the injection of siRNA-LNP. This work is able to demonstrate significantly increased hepatic uptake of siRNA-LNP by the BLT1i in Ldlr-/- mice by in vivo imaging and discover an induction of specific uptake-related proteins. Irrespective of the inhibitors and Ldlr deficiency, the siRNA-LNP induced RNA-binding and transport-related proteins in liver, including haptoglobin (HP) that is also identified as most upregulated serum protein. This work observes a downregulation of proteins functioning in hepatic detoxification and of serum opsonins. Most strikingly, the HDLRi reduces hepatic uptake and increases siRNA accumulation in spleen and myeloid immune cells of blood and liver. RNA sequencing demonstrates leukocyte recruitment by the siRNA-LNP and the HDLRi through induction of chemokine ligands in liver tissue. The data provide insights into key mechanisms of siRNA-LNP biodistribution and indicate that the HDLRi has potential for extrahepatic and leukocyte targeting.

Xestospongia muta Fraction-7 and Linoleic Acid: Effects on SR-BI Gene Expression and HDL Cholesterol Uptake

Xestospongia muta is a marine sponge belonging to the family Petrosiidae. It is an important source of biologically active marine natural products, with different kinds of essential fatty acids. Scavenger receptor class B type I (SR-BI) is the main receptor for high-density lipoprotein (HDL) cholesterol, which plays a pivotal role in preventing atherosclerosis. It removes cholesterol from HDL cholesterol, returning lipid-poor lipoprotein into blood circulation. The present study investigated the effects of X. muta Fraction-7 and linoleic acid on SR-BI gene expression and HDL cholesterol uptake. In vitro studies of the activity of X. muta and linoleic acid against the therapeutic target for hypercholesterolemia were conducted using the HDL receptor SR-BI via luciferase assay and HepG2 cells. In the present study, Fraction-7 of X. muta showed the highest expression level of the SR-BI gene via luciferase assay. Profiling of Fraction-7 of X. muta by GC-MS revealed 58 compounds, comprising various fatty acids, particularly linoleic acid. The in vitro study in HepG2 cells showed that the Fraction-7 of X. muta and linoleic acid (an active compound in X. muta) increased SR-BI mRNA expression by 129% and 85%, respectively, compared to the negative control. Linoleic acid increased HDL uptake by 3.21-fold compared to the negative control. Thus, the Fraction-7 of X. muta and linoleic acid have the potential to be explored as adjuncts in the treatment of hypercholesterolemia to prevent or reduce the severity of atherosclerosis development.

Synthetic Lipid Nanoparticles Actively Target Acute Myeloid Leukemia (AML) Cells and Induce Ferroptosis through Decreased Expression of Glutathione Peroxidase 4

Introduction: Relapsed and refractory AML patients have a 30% 5-year overall survival (SEER 2012-2018). We found that certain cancer cells that are highly dependent on cholesterol uptake and express the high-affinity receptor for cholesterol-rich high-density lipoproteins (HDL), scavenger receptor type B-1 (SR-B1), also highly express the redox enzyme, glutathione peroxidase 4 (GPX4) (Rink et al 2020). GPX4 has been shown to detoxify lipid peroxides in cancer cells and prevent cell death through ferroptosis, an iron-dependent redox form of cell death. Using a synthetic lipid nanoparticle (Au-LNP) that actively targets SR-B1, we showed modulation of cancer cell cholesterol uptake and metabolism is accompanied by a reduction in GPX4 expression. As a result, Au-LNP therapy kills these cells via ferroptosis. In AML, high GPX4 expression is associated with worse overall survival (Akiyama et al. Blood 2021). Therefore, we hypothesized that high SR-B1 expression would enable Au-LNP to target AML cells, modulate cell cholesterol, and reduce GPX4 resulting in AML cell death by ferroptosis. Methods: GEPIA is an interactive web server that houses the RNA sequencing expression data from the TCGA and the GTEx projects. We selected the LAML database and interrogated the survival data using the top 25% and bottom 25% SR-B1 expression as cutoffs. The cell lines MV4-11 (biphenotypic B-myelomonocytic AML with translocation t(4;11) and FLT3-ITD mutation), HEL (erythroblast AML), and U937 (promonocytic AML) were used for these studies. Au-LNPs were synthesized by surface-functionalizing 5nm gold (Au) nanoparticles with apolipoprotein A-I and phospholipids. SR-B1 and GPX4 levels were measured by western blot and flow cytometry. Cell viability was measured using the MTS assay, annexin V/propidium iodide staining, and a colony forming assay. Colonies were counted by an automated microscope and analyzed with Fiji. Lipid oxidation levels were measured by a C11 BODIPY flow cytometry assay. Surface biomarker changes were measured by flow cytometry. Primary AML samples were obtained by collecting the buffy coat from peripheral blood samples from 2 patients with AML and isolating CD34 positive blasts (IRB# STU00208941). Results: In the LAML database in GEPIA, we found a significant survival detriment with high SR-B1 expression (75th percentile) when compared with low SR-B1 expression (25th percentile; p = 0.023; hazard ratio = 2.5) (Figure 1). SR-B1 expression was observed in all three AML cell lines tested (MV4-11, HEL, U937). Au-LNPs potently induced cell death in each cell line, with IC50s for MV4-11 (4.7 nM), HEL (0.8 nM), and U937 (4.6 nM) that were much lower than with cytarabine (535 nM, 259 nM, and 96 nM, respectively). In the colony forming assays, Au-LNPs significantly decreased MV4-11 colony counts compared with PBS control. By contrast, β-Hydroxy β-methylglutaryl-CoA (HMG-CoA) inhibitors, such as atorvastatin and rosuvastatin, had no effect on cell viability, with concentrations up to 50 µM. Au-LNPs reduced GPX4 expression in MV4-11 and HEL cells, leading to the accumulation of lipid peroxides by C11 BODIPY assay. Au-LNPs-induced cell death was prevented by the addition of the lipophilic antioxidant ferrostatin-1 and the iron chelator deferoxamine (DFO), supporting the hypothesis that Au-LNP-induced cell death occurred by ferroptosis. Low dose Au-LNPs increased expression of CD11c (dendritic cell marker), CD16 (monocyte marker), and CD24 (neutrophil marker) on MV4-11 cells, suggesting an increase in myeloid differentiation. Finally, we isolated CD34+ blasts from peripheral blood samples of two AML patients. Both patients’ blasts expressed SR-B1 as detected by flow cytometry, and Au-LNPs showed significant cell killing in both patient samples. Conclusion: We have demonstrated that Au-LNP target SR-B1, expressed by both AML cell lines and in primary AML samples, and induce cell death by ferroptosis resulting from depletion of GPX4. Future studies targeting the cholesterol scavenger receptor SR-B1 and investigating relevant resistance pathways and opportunities for synergy with available agents are indicated. Figure 1. Overall survival of AML patients in the LAML database stratified by scavenger receptor B1 expression. (Tang, Z. et al. (2017) GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res, 10.1093/nar/gkx247) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Implications of High-Density Cholesterol Metabolism for Oocyte Biology and Female Fertility.

Cholesterol is an essential component of animal cells. Different regulatory mechanisms converge to maintain adequate levels of this lipid because both its deficiency and excess are unfavorable. Low cell cholesterol content promotes its synthesis and uptake from circulating lipoproteins. In contrast, its excess induces the efflux to high-density lipoproteins (HDL) and their transport to the liver for excretion, a process known as reverse cholesterol transport. Different studies suggest that an abnormal HDL metabolism hinders female fertility. HDL are the only lipoproteins detected in substantial amounts in follicular fluid (FF), and their size and composition correlate with embryo quality. Oocytes obtain cholesterol from cumulus cells via gap junctions because they cannot synthesize cholesterol de novo and lack HDL receptors. Recent evidence has supported the possibility that FF HDL play a major role in taking up excess unesterified cholesterol (UC) from the oocyte. Indeed, genetically modified mouse models with disruptions in reverse cholesterol transport, some of which show excessive circulating UC levels, exhibit female infertility. Cholesterol accumulation can affect the egg´s viability, as reported in other cell types, and activate the plasma membrane structure and activity of membrane proteins. Indeed, in mice deficient for the HDL receptor Scavenger Class B Type I (SR-B1), excess circulating HDL cholesterol and UC accumulation in oocytes impairs meiosis arrest and hinders the developmental capacity of the egg. In other cells, the addition of cholesterol activates calcium channels and dysregulates cell death/survival signaling pathways, suggesting that these mechanisms may link altered HDL cholesterol metabolism and infertility. Although cholesterol, and lipids in general, are usually not evaluated in infertile patients, one study reported high circulating UC levels in women showing longer time to pregnancy as an outcome of fertility. Based on the evidence described above, we propose the existence of a well-regulated and largely unexplored system of cholesterol homeostasis controlling traffic between FF HDL and oocytes, with significant implications for female fertility.

Cholesterol supports bovine granulosa cell inflammatory responses to lipopolysaccharide.

Bovine granulosa cells need to be cultured with serum to generate inflammation in response to bacterial lipopolysaccharide. This study shows that it is cholesterol that facilitates this lipopolysaccharide-stimulated cytokine secretion. During bacterial infections of the bovine uterus or mammary gland, ovarian granulosa cells mount inflammatory responses to lipopolysaccharide (LPS). In vitro, LPS stimulates granulosa cell secretion of the cytokines IL-1α and IL-1β and the chemokine IL-8. These LPS-stimulated inflammatory responses depend on culturing granulosa cells with serum, but the mechanism is unclear. Here, we tested the hypothesis that cholesterol supports inflammatory responses to LPS in bovine granulosa cells. We used granulosa cells isolated from 4 to 8 mm and >8.5 mm diameter ovarian follicles and manipulated the availability of cholesterol. We found that serum or follicular fluid containing cholesterol increased LPS-stimulated secretion of IL-1α and IL-1β from granulosa cells. Conversely, depleting cholesterol using methyl-β-cyclodextrin diminished LPS-stimulated secretion of IL-1α, IL-1β and IL-8 from granulosa cells cultured in serum. Follicular fluid contained more high-density lipoprotein cholesterol than low-density lipoprotein cholesterol, and granulosa cells expressed the receptor for high-density lipoprotein, scavenger receptor class B member 1 (SCARB1). Furthermore, culturing granulosa cells with high-density lipoprotein cholesterol, but not low-density lipoprotein or very low-density lipoprotein cholesterol, increased LPS-stimulated inflammation in granulosa cells. Cholesterol biosynthesis also played a role in granulosa cell inflammation because RNAi of mevalonate pathway enzymes inhibited LPS-stimulated inflammation. Finally, treatment with follicle-stimulating hormone, but not luteinising hormone, increased LPS-stimulated granulosa cell inflammation, and follicle-stimulating hormone increased SCARB1 protein. However, changes in inflammation were not associated with changes in oestradiol or progesterone secretion. Taken together, these findings imply that cholesterol supports inflammatory responses to LPS in granulosa cells.

Decoding Functional High-Density Lipoprotein Particle Surfaceome Interactions.

High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport (RCT) and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the molecular level. During HDL’s journey throughout the body, its functions are mediated through interactions with cell surface receptors on different cell types. To characterize and better understand the functional interplay between HDL particles and tissue, we analyzed the surfaceome-residing receptor neighborhoods with which HDL potentially interacts. We applied a combination of chemoproteomic technologies including automated cell surface capturing (auto-CSC) and HATRIC-based ligand–receptor capturing (HATRIC-LRC) on four different cellular model systems mimicking tissues relevant for RCT. The surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAECs) revealed the main currently known HDL receptor scavenger receptor B1 (SCRB1), as well as 155 shared cell surface receptors representing potential HDL interaction candidates. Since vascular endothelial growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF-modulated surfaceome of HAEC using the auto-CSC technology. VEGF-A treatment led to the remodeling of the surfaceome of HAEC cells, including the previously reported higher surfaceome abundance of SCRB1. In total, 165 additional receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated receptors potentially involved in HDL function. Using the HATRIC-LRC technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor tyrosine-protein kinase Mer (MERTK). Through RNA interference, we revealed its contribution to endothelial HDL binding and uptake. Furthermore, subsequent proximity ligation assays (PLAs) demonstrated the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence binding and uptake of HDL.