Intracellular Cholesterol Accumulation Research Articles

Coordination of oxysterol binding protein 1 and VAP-A/B modulates the generation of cholesterol and viral inclusion bodies to promote grass carp reovirus replication.

Similar to other RNA viruses, grass carp reovirus, the causative agent of the hemorrhagic disease, replicates in cytoplasmic viral inclusion bodies (VIBs), orchestrated by host proteins and lipids. The host pathways that facilitate the formation and function of GCRV VIBs are poorly understood. This work demonstrates that GCRV manipulates grass carp oxysterol binding protein 1 (named as gcOSBP1) and vesicle-associated membrane protein-associated protein A/B (named as gcVAP-A/B), 3 components of cholesterol transport pathway, to generate VIBs. By siRNA-mediated knockdown, we demonstrate that gcOSBP1 is an essential host factor for GCRV replication. We reveal that the nonstructural proteins NS80 and NS38 of GCRV interact with gcOSBP1, and that the gcOSBP1 is recruited by NS38 and NS80 for promoting the generation of VIBs. gcOSBP1 increases the expression of gcVAP-A/B and promotes the accumulation of intracellular cholesterol. gcOSBP1 also interacts with gcVAP-A/B for forming gcOSBP1-gcVAP-A/B complexes, which contribute to enhance the accumulation of intracellular cholesterol and gcOSBP1-mediated generation of VIBs. Inhibiting cholesterol accumulation by lovastatin can completely abolish the effects of gcOSBP1 and/or gcVAP-A/B in promoting GCRV infection, suggesting that cholesterol accumulation is vital for gcOSBP1- and/or gcVAP-A/B-mediated GCRV replication. Thus, our results, which highlight that gcOSBP1 functions in the replication of GCRV via its interaction with essential viral proteins for forming VIBs and with host gcVAP-A/B, provide key molecular targets for obtaining anti-hemorrhagic disease grass carp via gene editing technology.

#1071 The effect and mechanism of SGLT2 inhibitor on improving inflammatory injury of glomerular endothelial cells in diabetes

Abstract Background and Aims ATP-binding cassette transporter A1 (ABCA1), which reduces intracellular cholesterol accumulation, is highly expressed in the kidney. Studies have shown that sodium-glucose co-transporter 2 inhibitors (SGLT2) inhibitors promote ABCA1 expression in multiple tissues and organs. However, the effect of SGLT2 inhibitors on ABCA1 expression in diabetic kidney disease (DKD) is unclear. In this study, the effect of empagliflozin, an SGLT2 inhibitor, on ABCA1 in glomerular endothelial cells(GECs) of DKD and the possible mechanisms linking ABCA1 to GECs inflammatory injury were investigated. Method Kidney tissues were obtained from renal biopsies of patients with early and advanced DKD. db/db mice were used as animal models of type 2 diabetes and the effects of SGLT2 inhibitors on ABCA1 were analyzed by treatment with empagliflozin. The effects of ABCA1 overexpression on glomerular lipid deposition and kidney injury were examined in a type 2 diabetic mouse model with ABCA1 overexpression (db/db + ABCA1 mice), and human renal glomerular endothelial cells (HRGECs) cultured in high glucose and high cholesterol conditions, which simulated type 2 diabetes in vitro. Results The expression of ABCA1 in the glomeruli of DKD patients and db/db mice was significantly decreased. Enhancing ABCA1 expression in the kidney tissue of diabetic mice by adenovirus transduction improved renal cholesterol deposition, inhibited the overactivation of C3a/C5a, and improved renal inflammatory injury. In addition, in cultured HRGECs under high glucose and high cholesterol conditions, ABCA1 deficiency increased the deposit of cellular cholesterol, contributed to overactivation of C3a/C5a, and induced inflammatory injury. Overexpression of ABCA1 enhancing cholesterol efflux or inhibition of C3a/C5a activation in vitro significantly protected against glomerular endothelial injury stimulated by high glucose and high cholesterol. Moreover, SGLT2 inhibitor empagliflozin up-regulated the expression of ABCA1 and down-regulated the levels of cholesterol, C3a/C5a and inflammatory cytokines in diabetic mice and HRGECs. Conclusion SGLT2 inhibitor (empagliflozin) might play a renal protective role by up-regulating ABCA1-mediated cholesterol outflow, inhibiting the activation of C3a/C5a, and then ameliorating the inflammatory injury of GECs.

Cholesterol Modulation Attenuates the AD-like Phenotype Induced by Herpes Simplex Virus Type 1 Infection.

Cholesterol, a crucial component of cell membranes, influences various biological processes, including membrane trafficking, signal transduction, and host-pathogen interactions. Disruptions in cholesterol homeostasis have been linked to congenital and acquired conditions, including neurodegenerative disorders such as Alzheimer's disease (AD). Previous research from our group has demonstrated that herpes simplex virus type I (HSV-1) induces an AD-like phenotype in several cell models of infection. This study explores the interplay between cholesterol and HSV-1-induced neurodegeneration. The impact of cholesterol was determined by modulating its levels with methyl-beta-cyclodextrin (MβCD) using the neuroblastoma cell lines SK-N-MC and N2a. We have found that HSV-1 infection triggers the intracellular accumulation of cholesterol in structures resembling endolysosomal/autophagic compartments, a process reversible upon MβCD treatment. Moreover, MβCD exhibits inhibitory effects at various stages of HSV-1 infection, underscoring the importance of cellular cholesterol levels, not only in the viral entry process but also in subsequent post-entry stages. MβCD also alleviated several features of AD-like neurodegeneration induced by viral infection, including lysosomal impairment and intracellular accumulation of amyloid-beta peptide (Aβ) and phosphorylated tau. In conclusion, these findings highlight the connection between cholesterol, neurodegeneration, and HSV-1 infection, providing valuable insights into the underlying mechanisms of AD.

Presenilin Deficiency Results in Cellular Cholesterol Accumulation by Impairment of Protein Glycosylation and NPC1 Function.

Presenilin proteins (PS1 and PS2) represent the catalytic subunit of γ-secretase and play a critical role in the generation of the amyloid β (Aβ) peptide and the pathogenesis of Alzheimer disease (AD). However, PS proteins also exert multiple functions beyond Aβ generation. In this study, we examine the individual roles of PS1 and PS2 in cellular cholesterol metabolism. Deletion of PS1 or PS2 in mouse models led to cholesterol accumulation in cerebral neurons. Cholesterol accumulation was also observed in the lysosomes of embryonic fibroblasts from Psen1-knockout (PS1-KO) and Psen2-KO (PS2-KO) mice and was associated with decreased expression of the Niemann-Pick type C1 (NPC1) protein involved in intracellular cholesterol transport in late endosomal/lysosomal compartments. Mass spectrometry and complementary biochemical analyses also revealed abnormal N-glycosylation of NPC1 and several other membrane proteins in PS1-KO and PS2-KO cells. Interestingly, pharmacological inhibition of N-glycosylation resulted in intracellular cholesterol accumulation prominently in lysosomes and decreased NPC1, thereby resembling the changes in PS1-KO and PS2-KO cells. In turn, treatment of PS1-KO and PS2-KO mouse embryonic fibroblasts (MEFs) with the chaperone inducer arimoclomol partially normalized NPC1 expression and rescued lysosomal cholesterol accumulation. Additionally, the intracellular cholesterol accumulation in PS1-KO and PS2-KO MEFs was prevented by overexpression of NPC1. Collectively, these data indicate that a loss of PS function results in impaired protein N-glycosylation, which eventually causes decreased expression of NPC1 and intracellular cholesterol accumulation. This mechanism could contribute to the neurodegeneration observed in PS KO mice and potentially to the pathogenesis of AD.

Abstract 3046: Macrophage-smooth Muscle Cell Interactions In Atherosclerotic Foam Cell Formation

Background: A hallmark of atherosclerosis is uncontrolled uptake of atherogenic lipoproteins by artery wall cells that accumulate large amounts of cholesteryl esters (“foam cells”). While smooth muscle cells (SMCs) and macrophages are in close proximity in lesions and contribute to cholesterol accumulation, whether these cells influence each other’s tendency to form foam cells is not clearly established. Hypothesis: Direct and indirect interactions between SMCs and macrophages promote foam cell formation by both cell types. Method: Human vascular SMCs were seeded onto macrophages to mimic direct macrophage-SMC interactions (n=5). Alternatively, conditioned media generated from human macrophages or SMCs with or without lipoprotein exposure was incubated with the alternate cell type (n=3). Aggregated lipoproteins were added in both models to induce cellular cholesterol accumulation. Intracellular cholesterol was quantified to compare lipoprotein uptake in cocultures relative to monocultures. Crosstalk based on CellChat analysis using single-cell RNA sequencing data from human coronary samples was performed to identify potential factors responsible for foam cell development (n=3). One-way ANOVA was used for statistical analysis. Result: SMCs showed a 4-fold (+/-0.3, p=0.002) increase in cell cholesterol accumulation when in direct contact with macrophages and a 3-fold (+/- 0.3, p<0.0001) increase when exposed to the conditioned media from macrophage foam cells. In contrast, macrophages showed a 3-fold reduction (+/- 0.3, p=0.001) in intracellular cholesterol accumulation when in direct contact with SMCs and a 2-fold reduction (+/-0.2, p=0.0005) when exposed to SMC foam cell-conditioned media. CellChat analysis suggested macrophage foam cells but not macrophage non-foam cells promote SMC foam cell formation by secretion of inflammatory cytokines, and that SMCs promote macrophage foam cell formation by secretion of extracellular matrix components. Conclusion: Our study provides novel understanding of how macrophage-SMC interactions influence macrophage and SMC foam cell formation. Further studies will be presented examining the role of SMC extracellular matrix proteins and macrophage-secreted cytokines on foam cell formation.

Towards a Unitary Hypothesis of Alzheimer's Disease Pathogenesis.

The "amyloid cascade" hypothesis of Alzheimer's disease (AD) pathogenesis invokes the accumulation in the brain of plaques (containing the amyloid-β protein precursor [AβPP] cleavage product amyloid-β [Aβ]) and tangles (containing hyperphosphorylated tau) as drivers of pathogenesis. However, the poor track record of clinical trials based on this hypothesis suggests that the accumulation of these peptides is not the only cause of AD. Here, an alternative hypothesis is proposed in which the AβPP cleavage product C99, not Aβ, is the main culprit, via its role as a regulator of cholesterol metabolism. C99, which is a cholesterol sensor, promotes the formation of mitochondria-associated endoplasmic reticulum (ER) membranes (MAM), a cholesterol-rich lipid raft-like subdomain of the ER that communicates, both physically and biochemically, with mitochondria. We propose that in early-onset AD (EOAD), MAM-localized C99 is elevated above normal levels, resulting in increased transport of cholesterol from the plasma membrane to membranes of intracellular organelles, such as ER/endosomes, thereby upregulating MAM function and driving pathology. By the same token, late-onset AD (LOAD) is triggered by any genetic variant that increases the accumulation of intracellular cholesterol that, in turn, boosts the levels of C99 and again upregulates MAM function. Thus, the functional cause of AD is upregulated MAM function that, in turn, causes the hallmark disease phenotypes, including the plaques and tangles. Accordingly, the MAM hypothesis invokes two key interrelated elements, C99 and cholesterol, that converge at the MAM to drive AD pathogenesis. From this perspective, AD is, at bottom, a lipid disorder.

Cholesterol metabolism: physiological versus pathological aspects in intracerebral hemorrhage.

Cholesterol is an important component of plasma membranes and participates in many basic life functions, such as the maintenance of cell membrane stability, the synthesis of steroid hormones, and myelination. Cholesterol plays a key role in the establishment and maintenance of the central nervous system. The brain contains 20% of the whole body's cholesterol, 80% of which is located within myelin. A huge number of processes (e.g., the sterol regulatory element-binding protein pathway and liver X receptor pathway) participate in the regulation of cholesterol metabolism in the brain via mechanisms that include cholesterol biosynthesis, intracellular transport, and efflux. Certain brain injuries or diseases involving crosstalk among the processes above can affect normal cholesterol metabolism to induce detrimental consequences. Therefore, we hypothesized that cholesterol-related molecules and pathways can serve as therapeutic targets for central nervous system diseases. Intracerebral hemorrhage is the most severe hemorrhagic stroke subtype, with high mortality and morbidity. Historical cholesterol levels are associated with the risk of intracerebral hemorrhage. Moreover, secondary pathological changes after intracerebral hemorrhage are associated with cholesterol metabolism dysregulation, such as neuroinflammation, demyelination, and multiple types of programmed cell death. Intracellular cholesterol accumulation in the brain has been found after intracerebral hemorrhage. In this paper, we review normal cholesterol metabolism in the central nervous system, the mechanisms known to participate in the disturbance of cholesterol metabolism after intracerebral hemorrhage, and the links between cholesterol metabolism and cell death. We also review several possible and constructive therapeutic targets identified based on cholesterol metabolism to provide cholesterol-based perspectives and a reference for those interested in the treatment of intracerebral hemorrhage.

Fatostatin promotes anti-tumor immunity by reducing SREBP2 mediated cholesterol metabolism in tumor-infiltrating T lymphocytes

Aberrant lipid metabolism impacts intratumoral T cell-mediated immune response and tumor growth. Fatostatin functions as an inhibitor of sterol regulatory element binding protein (SREBP) activation. However, the complex effects of fatostatin on cholesterol metabolism in the tumor microenvironment (TME) and its influence on T cell anti-tumor immunity remain unclear. In this study, fatostatin effectively suppressed B16 melanoma, MC38 colon cancer, and Lewis lung cancer (LLC) transplanted tumor growth in immunocompetent mice by reducing SREBPs-mediated lipid metabolism, especially cholesterol levels. Mechanistically, fatostatin decreased intracellular cholesterol accumulation and inhibited X-box binding protein 1 (XBP1)-mediated endoplasmic reticulum (ER) stress, reducing Treg cells and alleviating CD8+ T cell exhaustion in the TME, exerting anti-tumor activity. Nevertheless， this effect was impaired in immunodeficient nude mice, suggesting fatostatin's anti-tumor efficacy in transplanted tumors partly relies on T cell-mediated anti-tumor immunity. Our study highlights SREBP2-mediated cholesterol metabolism as a potential strategy for anti-tumor immunotherapy, and confirms fatostatin's promise in tumor immunotherapy.

Abstract 5286: M2 to M1 repolarization of tumor-associated macrophages via the lysosomal effect of itraconazole

Abstract Objective: Itraconazole (ITZ), an antifungal agent, has been reported to have tumor agnostic anticancer effects. Previous reports have shown prolonged survival in patients with lung and ovarian cancer who received ITZ. The anticancer mechanisms vary depending on the cancer type and stromal cells. Based on our previous studies, we focused on tumor-associated macrophages (TAMs). Methods: Anti-tumorigenic M1 and pro-tumorigenic M2 macrophages were established from THP-1 cells and their phenotypes were determined based on morphology, cell surface antigens by Western blotting, and secreted proteins by ELISA. Bulk proteomic analysis of cell proteins was conducted using liquid chromatography-tandem mass spectrometry. The viability of CaSki cervical cancer cells was evaluated both in culture with the supernatant and co-culture with M2 macrophages after treatment with ITZ (10-5 M). Single-cell RNA sequencing (scRNA-seq) with surface labeling of M2 macrophages with and without the ITZ was performed. Using LoupeBrowser (10x Genomics, Inc.), ITZ-treated M2 macrophages were divided into two groups, one with newly emerged clusters after ITZ treatment (group 1) and the other (group 2). Differentially expressed genes between group 1 and group 2 were identified based on a two-fold and a 0.05 adjusted p value and were subjected to Reactome pathway analysis. Significant pathways were identified based on both p value < 0.05 and FDR <0.3. A triple-labeling immunofluorescence study was conducted to detect cholesterol, IL-1β, and organelles (the endoplasmic reticulum, phagosome, and lysosome). Results: ITZ changed M2 macrophages to M1-like morphology and protein expression. While co-culture with M2 macrophages promoted cancer cell proliferation, both culture with the supernatant and co-culture with ITZ-treated M2 macrophages significantly inhibited CaSki cell growth. scRNA-seq identified newly emerged clusters (group 1) among M2 macrophages following ITZ treatment. Group 1 had elevated mRNA expression of M1 markers, including IL1β, IL6, CD86, TL4, GAPDH, and RKM, compared to group 2. Group 1 expressed the CD86 and TLR4 surface antigen proteins. Pathway analysis identified 176 significant pathways including lysosome vesicle biogenesis. Immunofluorescence analysis showed that M1-like macrophages in ITZ-treated M2 macrophages had enlarged lysosomes containing cholesterol. Conclusion: ITZ repolarized THP-1 derived M2 macrophage to an M1-like phenotype. The mechanism of repolarization involves intracellular cholesterol accumulation via lysosomal dysfunction. Citation Format: Yumi Takimoto, Hiroshi Tsubamoto, Kazuko Sakata, Roze Isono-Taniguchi, Tomoko Ueda, Hiroaki Shibahara. M2 to M1 repolarization of tumor-associated macrophages via the lysosomal effect of itraconazole [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5286.

Targeted Delivery of Mesenchymal Stem Cell-Derived Bioinspired Exosome-Mimetic Nanovesicles with Platelet Membrane Fusion for Atherosclerotic Treatment.

Accumulating evidence indicates that mesenchymal stem cells (MSCs)-derived exosomes hold significant potential for the treatment of atherosclerosis. However, large-scale production and organ-specific targeting of exosomes are still challenges for further clinical applications. This study aims to explore the targeted efficiency and therapeutic potential of biomimetic platelet membrane-coated exosome-mimetic nanovesicles (P-ENVs) in atherosclerosis. To produce exosome-mimetic nanovesicles (ENVs), MSCs were successively extruded through polycarbonate porous membranes. P-ENVs were engineered by fusing MSC-derived ENVs with platelet membranes and characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. The stability and safety of P-ENVs were also assessed. The targeted efficacy of P-ENVs was evaluated using an in vivo imaging system (IVIS) spectrum imaging system and immunofluorescence. Histological analyses, Oil Red O (ORO) staining, and Western blot were used to investigate the anti-atherosclerotic effectiveness of P-ENVs. Both ENVs and P-ENVs exhibited similar characteristics to exosomes. Subsequent miRNA sequencing of P-ENVs revealed their potential to mitigate atherosclerosis by influencing biological processes related to cholesterol metabolism. In an ApoE-/- mice model, the intravenous administration of P-ENVs exhibited enhanced targeting of atherosclerotic plaques, resulting in a significant reduction in lipid deposition and necrotic core area. Our in vitro experiments showed that P-ENVs promoted cholesterol efflux and reduced total cholesterol content in foam cells. Further analysis revealed that P-ENVs attenuated intracellular cholesterol accumulation by upregulating the expression of the critical cholesterol transporters ABCA1 and ABCG1. This study highlighted the potential of P-ENVs as a novel nano-drug delivery platform for enhancing drug delivery efficiency while concurrently mitigating adverse reactions in atherosclerotic therapy.

Cav3.1 T-type calcium channel blocker NNC 55-0396 reduces atherosclerosis by increasing cholesterol efflux

Calcium channel blockers (CCBs) are commonly used as antihypertensive agents. While certain L-type CCBs exhibit antiatherogenic effects, the impact of Cav3.1 T-type CCBs on antiatherogenesis and lipid metabolism remains unexplored. NNC 55–0396 (NNC) is a highly selective blocker of T-type calcium channels (Cav3.1 channels). We investigated the effects of NNC on relevant molecules and molecular mechanisms in human THP-1 macrophages. Cholesterol efflux, an indicator of reverse cholesterol transport (RCT) efficiency, was assessed using [3H]-labeled cholesterol. In vivo, high cholesterol diet (HCD)-fed LDL receptor knockout (Ldlr-/-) mice, an atherosclerosis-prone model, underwent histochemical staining to analyze plaque burden. Treatment of THP-1 macrophages with NNC facilitated cholesterol efflux and reduced intracellular cholesterol accumulation. Pharmacological and genetic interventions demonstrated that NNC treatment or Cav3.1 knockdown significantly enhanced the protein expression of scavenger receptor B1 (SR-B1), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and liver X receptor alpha (LXRα) transcription factor. Mechanistic analysis revealed that NNC activates p38 and c-Jun N-terminal kinase (JNK) phosphorylation, leading to increased expression of ABCA1, ABCG1, and LXRα-without involving the microRNA pathway. LXRα isrequired for NNC–induced ABCA1 and ABCG1 expression. Administering NNC diminished atherosclerotic lesion area and lipid deposition in HCD-fed Ldlr-/- mice. NNC's anti-atherosclerotic effects, achieved through enhanced cholesterol efflux and inhibition of lipid accumulation, suggest a promising therapeutic approach for hypertensive patients with atherosclerosis. This research highlights the potential of Cav3.1 T-type CCBs in addressing cardiovascular complications associated with hypertension.

Evidence for the Involvement of Gene Regulation of Inflammatory Molecules in the Accumulation of Intracellular Cholesterol: The Mechanism of Foam Cell Formation in Atherosclerosis.

The relationship between the cellular pro-inflammatory response and intracellular lipid accumulation in atherosclerosis is not sufficiently studied. Transcriptomic analysis is one way to establish such a relationship. Previously, we identified 10 potential key genes (IL-15, CXCL8, PERK, IL-7, IL-7R, DUSP1, TIGIT, F2RL1, TSPYL2, and ANXA1) involved in cholesterol accumulation in macrophages. It should be noted that all these genes do not directly participate in cholesterol metabolism, but encode molecules related to inflammation. In this study, we conducted a knock-down of the 10 identified key genes using siRNA to determine their possible role in cholesterol accumulation in macrophages. To assess cholesterol accumulation, human monocyte-derived macrophages (MDM) were incubated with atherogenic LDL from patients with atherosclerosis. Cholesterol content was assessed by the enzymatic method. Differentially expressed genes were identified with DESeq2 analysis. Master genes were determined by the functional analysis. We found that only 5 out of 10 genes (IL-15, PERK, IL-7, IL-7R, ANXA1) can affect intracellular lipid accumulation. Knock-down of the IL-15, PERK, and ANXA1 genes prevented lipid accumulation, while knock-down of the IL-7 and IL-7R genes led to increased intracellular lipid accumulation during incubation of MDM with atherogenic LDL. Seventeen overexpressed genes and 189 underexpressed genes were obtained in the DGE analysis, which allowed us to discover 20 upregulated and 86 downregulated metabolic pathways, a number of which are associated with chronic inflammation and insulin signaling. We also elucidated 13 master regulators of cholesterol accumulation that are immune response-associated genes. Thus, it was discovered that 5 inflammation-related master regulators may be involved in lipid accumulation in macrophages. Therefore, the pro-inflammatory response of macrophages may trigger foam cell formation rather than the other way around, where intracellular lipid accumulation causes an inflammatory response, as previously assumed.

Evidence for hypoxia-induced dysregulated cholesterol homeostasis in preeclampsia: Insights into the mechanisms from human placental cells and tissues.

Preeclampsia (PE) poses a considerable risk to the long-term cardiovascular health of both mothers and their offspring due to a hypoxic environment in the placenta leading to reduced fetal oxygen supply. Cholesterol is vital for fetal development by influencing placental function. Recent findings suggest an association between hypoxia, disturbed cholesterol homeostasis, and PE. This study investigates the influence of hypoxia on placental cholesterol homeostasis. Using primary human trophoblast cells and placentae from women with PE, various aspects of cholesterol homeostasis were examined under hypoxic and hypoxia/reoxygenation (H/R) conditions. Under hypoxia and H/R, intracellular total and non-esterified cholesterol levels were significantly increased. This coincided with an upregulation of HMG-CoA-reductase and HMG-CoA-synthase (key genes regulating cholesterol biosynthesis), and a decrease in acetyl-CoA-acetyltransferase-1 (ACAT1), which mediates cholesterol esterification. Hypoxia and H/R also increased the intracellular levels of reactive oxygen species and elevated the expression of hypoxia-inducible factor (HIF)-2α and sterol-regulatory-element-binding-protein (SREBP) transcription factors. Additionally, exposure of trophoblasts to hypoxia and H/R resulted in enhanced cholesterol efflux to maternal and fetal serum. This was accompanied by an increased expression of proteins involved in cholesterol transport such as the scavenger receptor class B type I (SR-BI) and the ATP-binding cassette transporter G1 (ABCG1). Despite these metabolic alterations, mitogen-activated-protein-kinase (MAPK) signaling, a key regulator of cholesterol homeostasis, was largely unaffected. Our findings indicate dysregulation of cholesterol homeostasis at multiple metabolic points in both the trophoblast hypoxia model and placentae from women with PE. The increased cholesterol efflux and intracellular accumulation of non-esterified cholesterol may have critical implications for both the mother and the fetus during pregnancy, potentially contributing to an elevated cardiovascular risk later in life.

The role of STAT3/VAV3 in glucolipid metabolism during the development of HFD-induced MAFLD.

Metabolic-associated fatty liver disease (MAFLD) is a globally prevalent chronic hepatic disease. Previous studies have indicated that the activation of the signal transducer and activator of transcription3 (STAT3) plays a vital role in MAFLD progression at the very beginning. However, the specific association between STAT3 and abnormal hepatic metabolism remains unclear. In this study, activated inflammation was observed to induce abnormal glucolipid metabolic disorders in the hepatic tissues of high-fat diet (HFD)-fed ApoE-/- mice. Furthermore, we found that the activation of STAT3 induced by HFD might function as a transcriptional factor to suppress the expression of VAV3, which might participate in intracellular glucolipid metabolism and the regulation of glucose transporter 4 (GLUT4) storage vesicle traffic in the development of MAFLD both in vitro and in vivo. We verified that VAV3 deficiency could retard the GLUT4 membrane translocation and impair the glucose homeostasis. Additionally, VAV3 participates in cholesterol metabolism in hepatocytes, eventually resulting in the accumulation of intracellular cholesterol. Moreover, rAAV8-TBG-VAV3 was conducted to restore the expression of VAV3 in HFD-fed ApoE-/- mice. VAV3 overexpression was observed to improve glucose homeostasis as well as attenuate hepatic cholesterol accumulation in vivo. In conclusion, the STAT3/VAV3 signaling pathway might play a significant role in MAFLD by regulating glucose and cholesterol metabolism, and VAV3 might be a potential therapeutic strategy which could consequently ameliorate MAFLD.

Coffee pulp simulated digestion enhances its in vitro ability to decrease emulsification and digestion of fats, and attenuates lipid accumulation in HepG2 cell model

The coffee industry produces a considerable quantity of coffee pulp (CP), a by-product with high levels of caffeine, phenolic compounds, and dietary fiber, which are reportedly involved in the lipid homeostasis regulation required to maintain human health. This work's objective was to evaluate the hypolipidemic activity of coffee pulp flour (CPF) and aqueous extract (CPE) after static simulated digestion by the assessment of their in vitro capacity to decrease emulsification and digestion of fats, and lipid-lowering capacity in HepG2 cells after the induction of intracellular fat accumulation. The CPF and CPE digested fractions displayed in vitro hypolipidemic properties by preserving the reduction of micellar cholesterol solubility (27–34%) and the secondary bile acid-binding capacity (22–30%), increasing their primary bile acid-binding ability (2.7-fold and 2.4-fold, respectively), and inhibiting the lipase and the HMGCR (77–79% and 36–85%, respectively) activities. Moreover, the hypolipidemic properties of non-digested fractions enhanced the CPF potential to decrease lipid absorption. Both ingredients (CPF and CPE) demonstrated lipid-lowering effects since they effectively counteract the accumulation of intracellular triglycerides and cholesterol triggered by palmitic acid in hepatic cells after the simulated digestion. This study suggests that phenolic compounds, caffeine, and dietary fiber may be responsible for the lipid-lowering properties exhibited by the CP ingredients and their composition differences affect the above-mentioned properties exhibited in the simulated digestion. These results contribute to demonstrating that the CPF and the CPE may act as modulators of pathways involved in hepatic lipid accumulation and could be a key element in its prevention.

NPC1-like phenotype, with intracellular cholesterol accumulation and altered mTORC1 signaling in models of Parkinson's disease

Disruption of brain cholesterol homeostasis has been implicated in neurodegeneration. Nevertheless, the role of cholesterol in Parkinson's Disease (PD) remains unclear. We have used N2a mouse neuroblastoma cells and primary cultures of mouse neurons and 1-methyl-4-phenylpyridinium (MPP+), a known mitochondrial complex I inhibitor and the toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), known to trigger a cascade of events associated with PD neuropathological features. Simultaneously, we utilized other mitochondrial toxins, including antimycin A, oligomycin, and carbonyl cyanide chlorophenylhydrazone. MPP+ treatment resulted in elevated levels of total cholesterol and in a Niemann Pick type C1 (NPC1)-like phenotype characterized by accumulation of cholesterol in lysosomes. Interestingly, NPC1 mRNA levels were specifically reduced by MPP+. The decrease in NPC1 levels was also seen in midbrain and striatum from MPTP-treated mice and in primary cultures of neurons treated with MPP+. Together with the MPP+-dependent increase in intracellular cholesterol levels in N2a cells, we observed an increase in 5′ adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and a concomitant increase in the phosphorylated levels of mammalian target of rapamycin (mTOR). NPC1 knockout delayed cell death induced by acute mitochondrial damage, suggesting that transient cholesterol accumulation in lysosomes could be a protective mechanism against MPTP/MPP+ insult. Interestingly, we observed a negative correlation between NPC1 protein levels and disease stage, in human PD brain samples. In summary, MPP+ decreases NPC1 levels, elevates lysosomal cholesterol accumulation and alters mTOR signaling, adding to the existing notion that PD may rise from alterations in mitochondrial-lysosomal communication.

Cholesterol and matrisome pathways dysregulated by APOE4 in human glia

AbstractBackgroundThe association of Apolipoprotein E (APOE) ɛ4 risk with Alzheimer’s disease (AD) is well‐established. However, the impact of APOE ɛ4 on human brain cell function remains unclear. We hypothesized that human APOE ε4/ε4 (APOE 44) genotype contributes to disease risk through cell autonomous and non‐cell autonomous mechanisms in a human cell specific manner.MethodGlobal transcriptomic analyses were performed on astrocytes, microglia, mixed cortical cultures (neurons and astrocytes) and brain microvascular endothelial cells derived from human induced pluripotent stem cells (iPSCs) and post‐mortem AD brains. Lipid pathway dysregulation identified through GSEA, WGCNA and cell type deconvolution was validated in vitro using Gas Chromatography‐Mass Spectrometry (GC‐MS), filipin staining, autophagy fluorescent reporters with immunocytochemistry and western blotting to determine the subcellular compartment that accumulates cholesterol in iPSC‐derived astrocytes, and further confirmed by immunohistochemical analysis in human APOE4 post‐mortem brain. Matrisome pathway dysregulation was validated by Luminex multiplex immunoassays and intracellular protein levels in pure astrocytes and mixed cortical cultures.ResultGlobal transcriptomic analyses reveal that APOE 44 drives cholesterol metabolic dysregulation in astrocytes and microglia from human iPSCs and cell type‐deconvolved post‐mortem AD brain. APOE ε4 leads to elevated de novo cholesterol synthesis despite the intracellular accumulation of free cholesterol in lysosome and cholesterol ester in lipid droplets coupled with reduced efflux due to impaired lipophagy in iPSC‐derived astrocytes. Lysosomal cholesterol accumulation is found in astrocytes from APOE ε4 human brain. Activation of LXRs and induction of lipophagy ameliorates the lipid phenotypes in APOE ε4 astrocytes. Further, matrisome dysregulation is associated with upregulated chemotaxis, glial activation, and lipid biosynthesis in astrocytes co‐cultured with neurons, which recapitulates altered astrocyte matrisome signaling in human brain.ConclusionThus, human APOE ε4 causes human glia‐specific, cell autonomous and non‐cell autonomous cholesterol and matrisome pathway dysregulation that may increase AD risk.

Toxicological Mechanisms and Potencies of Organophosphate Esters in KGN Human Ovarian Granulosa Cells as Revealed by High-throughput Transcriptomics.

Despite the growing number of studies reporting potential risks associated with exposure to organophosphate esters (OPEs), their molecular mechanisms of action remain poorly defined. We used the high-throughput TempO-Seq™ platform to investigate the effects of frequently detected OPEs on the expression of ∼3000 environmentally responsive genes in KGN human ovarian granulosa cells. Cells were exposed for 48 h to one of five OPEs (0.1 to 50 μM): tris(methylphenyl) phosphate (TMPP), isopropylated triphenyl phosphate (IPPP), tert-butylphenyl diphenyl phosphate (BPDP), triphenyl phosphate (TPHP), or tris(2-butoxyethyl) phosphate (TBOEP). The sequencing data indicate that four OPEs induced transcriptional changes, whereas TBOEP had no effect within the concentration range tested. Multiple pathway databases were used to predict alterations in biological processes based on differentially expressed genes. At lower concentrations, inhibition of the cholesterol biosynthetic pathway was the predominant effect of OPEs; this was likely a consequence of intracellular cholesterol accumulation. At higher concentrations, BPDP and TPHP had distinct effects, primarily affecting pathways involved in cell cycle progression and other stress responses. Benchmark concentration (BMC) modelling revealed that BPDP had the lowest transcriptomic point of departure. However, in vitro to in vivo extrapolation modeling indicated that TMPP was bioactive at lower concentrations than the other OPEs. We conclude that these new approach methodologies provide information on the mechanism(s) underlying the effects of data-poor compounds and assist in the derivation of protective points of departure for use in chemical read-across and decision-making.

Anlotinib affects systemic lipid metabolism and induces lipid accumulation in human lung cancer cells

BackgroundAnlotinib has demonstrated encouraging clinical outcomes in the treatment of lung cancer, soft tissue sarcoma and thyroid carcinoma. Several clinical studies have shown a relationship between anlotinib treatment and the occurrence of hyperlipidemia. The fundamental mechanisms, however, are still largely unclear. Here, the effect of anlotinib on lipid metabolism in an animal model and human cancer cells was evaluated and the role of lipid metabolism in the antitumor efficacy of anlotinib was investigated.MethodsThe C57BL/6 J mouse model as well as A549 and H460 human lung cancer cell lines were used to examine the impact of anlotinib on lipid metabolism both in vivo and in vitro. Levels of triglycerides, high-density lipoprotein, low-density lipoprotein (LDL), and total cholesterol in serum or cell samples were determined using assay kits. The expression levels of crucial genes and proteins involved in lipid metabolism were measured by quantitative RT-PCR and Western blotting. Furthermore, exogenous LDL and knockdown of low-density lipoprotein receptor (LDLR) were used in H460 cells to investigate the relevance of lipid metabolism in the anticancer efficacy of anlotinib.ResultsAnlotinib caused hyperlipidemia in C57BL/6 J mice, possibly by downregulating hepatic LDLR-mediated uptake of LDL cholesterol. AMP-activated protein kinase and mammalian target of rapamycin inhibition may also be involved. Additionally, anlotinib enhanced sterol response element binding protein 1/2 nuclear accumulation as well as upregulated LDLR expression in A549 and H460 cells, which may be attributable to intracellular lipid accumulation. Knockdown of LDLR reduced intracellular cholesterol content, but interestingly, anlotinib significantly improved intracellular cholesterol accumulation in LDLR-knockdown cells. Both exogenous LDL and LDLR knockdown decreased the sensitivity of cells to anlotinib.ConclusionsAnlotinib modulates host lipid metabolism through multiple pathways. Anlotinib also exerts a significant impact on lipid metabolism in cancer cells by regulating key transcription factors and metabolic enzymes. In addition, these findings suggest lipid metabolism is implicated in anlotinib sensitivity.

Statins suppress cell-to-cell propagation of α-synuclein by lowering cholesterol

Cell-to-cell propagation of protein aggregates has been implicated in the progression of neurodegenerative diseases. However, the underlying mechanism and modulators of this process are not fully understood. Here, we screened a small-molecule library in a search for agents that suppress the propagation of α-synuclein and mutant huntingtin (mHtt). These screens yielded several molecules, some of which were effective against both α-synuclein and mHtt. Among these molecules, we focused on simvastatin and pravastatin. Simvastatin administration in a transgenic model of synucleinopathy effectively ameliorated behavioral deficits and α-synuclein accumulation, whereas pravastatin had no effect. Because only simvastatin enters the brain effectively, these results suggest that inhibition of brain cholesterol synthesis is important in simvastatin effects. In cultured cells, accumulation of intracellular cholesterol, induced by genetic ablation of the NPC1 gene or by pharmacological treatment with U18666A, increased α-synuclein aggregation and secretion. In contrast, lowering cholesterol using methyl-β-cyclodextrin or statins reversed α-synuclein aggregation and secretion in NPC1-knockout cells. Consistent with these observations, feeding a high-fat diet aggravated α-synuclein pathology and behavioral deficits in the preformed fibril-injected mouse model, an effect that was also reversed by simvastatin administration. These results suggest that statins suppress propagation of protein aggregates by lowering cholesterol in the brain.