Excessive Accumulation Of Cholesterol Research Articles

An update on ox-LDL-inducing vascular smooth muscle cell-derived foam cells in atherosclerosis.

Excess cholesterol accumulation induces the accumulation of foam cells, eventually accelerating atherosclerosis progress. Historically, the mechanisms of macrophage-derived foam cells have attracted attention because of their central role in plaque development, which was challenged by lineage tracing in union with single-cell sequencing (sc-seq). Accumulated studies have uncovered how vascular smooth muscle cells (VSMCs) proliferate and migrate to the vascular intima and accumulate, then transform into foam cells induced by surplus lipids, finally accounting for 30% to 70% of the total foam cells within the plaque of both mice and humans. Therefore, the mechanisms of VSMC-derived foam cells have received increasing attention. The review intends to summarize the transformation mechanism of VSMCs into foam cells induced by oxidized low-density lipoproteins (ox-LDL) in atherosclerosis.

Proatherogenic proteomic profile of LDL isolated from plasma of patients with diabetes mellitus: immunological aspects

Atherosclerosis is a vascular disease, which is based on a chronic inflammatory process, including complex interactions between blood components, as well as the cells that form the vessel wall, and lipid metabolism in general. Low-density lipoproteins (LDL) play a key role in pathogenesis of atherosclerosis. When the permeability of the endothelial layer of the vessel is impaired, LDL can penetrate into the intramural space and lead to excessive cholesterol accumulation by the cells from the intima subendothelial layer, leading to the onset of the inflammatory process and to the formation of foam cells, the main morphological component of the atherosclerotic plaque. However, the level of LDL in the patient’s blood plasma alone is not a key indicator of the development of atherosclerosis. Numerous studies point out the role of oxidized modifications of LDL (oxLDL) in the increased local accumulation of cholesterol in vascular wall cells, however, recent works have shown conflicting results regarding the role of oxLDL in the development of atherosclerosis. We hypothesize that other components of LDL may also influence the progression of atherosclerosis. It is a well-known fact that patients with diabetes mellitus (DM) suffer from cardiovascular diseases, in particular atherosclerosis, more often than patients without diagnosed DM and other autoimmune diseases, and the disease progresses faster. The purpose of this study was to identify risk biomarkers in LDL groups that indicate the relationship of the immune system with the development of atherosclerosis in such patients. LDL was isolated from patients and healthy donors using continuous ultracentrifugation with solutions of different densities, and LDL protein profile samples were measured using gas chromatography-mass spectrometry. We found 9 proteins that had a statistically significant difference between the samples (DM and control). In the diabetes group, the content of the antimicrobial peptide cathelicidin and lipopolysaccharide-binding protein was almost 2 times higher compared to the control. These proteins may be involved in the development of inflammation, leading to the progression of atherosclerosis. At the same time, a decrease in immunoglobulins and complement components (C9 and Complement C1s subcomponent) associated with LDL may contribute to the development of atherosclerosis.

7-Ketocholesterol promotes cholesterol uptake and inflammation in macrophage: Potential mechanisms of dietary COP in atherosclerosis promotion

This study investigated the effects and mechanisms of 7-ketocholesterol in promoting macrophage foaming and inflammation. Single-cell RNA sequencing showed that dietary cholesterol oxidation products increased the inflammatory macrophage and Trem2high macrophage numbers in the atherosclerotic aorta of ApoE−/− mice. Enrichment analysis demonstrated activation of inflammation-related pathways (NF-κB pathway and NLRP3 inflammasome) in inflammatory macrophages and cholesterol metabolism-related pathways (LDLR and ABCG1) in Trem2high macrophages. An oxidized-LDL-treated macrophage model was employed to verify the effects and mechanisms of 7-KC on macrophage inflammation and foam cell formation. 7-KC promoted the inflammation and M1 polarization of macrophage in association with the activations of the NF-κB pathway and NLRP3 inflammasome. Furthermore, 7-KC-mediated excessive cholesterol accumulation was attributed to enhanced cholesterol uptake rather than inhibition of cholesterol efflux. Consequently, LDLR expression was up-regulated by 7-KC, while no significant effect on ABCG1 expression was observed.

Umbilical Cord Mesenchymal Stem Cells Combined with Fufang Xueshuantong Capsule Attenuate Oxidative Stress and Vascular Lesions in Diabetic Rats by Activating Nrf-2/HO-1 Signaling Pathway.

Macrovascular lesions are the main cause of death and disability in diabetes mellitus, and excessive accumulation of cholesterol and lipids can lead to long-term and repeated damage of vascular endothelial cells. Umbilical cord mesenchymal stem cells (UCMSCs) can attenuate vascular endothelial damage in type 1 diabetic mice, while Fufang Xueshuantong capsule (FXC) has a protective effect on endothelial function; however, whether FXC in combination with UCMSCs can improve T2DM macrovascular lesions as well as its mechanism of action are not clear. Therefore, the aim of this study was to reveal the role of FXC + UCMSCs in T2DM vasculopathy and their potential mechanism in the treatment of T2DM. The control and T2DM groups were intragastrically administered with equal amounts of saline, the UCMSCs group was injected with UCMSCs (1×106, resuspended cells with 0.5 mL PBS) in the tail vein, the FXC group was intragastrically administered with 0.58 g/kg FXC, and the UCMSCs + FXC group was injected with UCMSCs (1×106) in the tail vein, followed by FXC (0.58 g/kg), for 8 weeks. We found that FXC+UCMSCs effectively reduced lipid levels (TG, TC, and LDL-C) and ameliorated aortic lesions in T2DM rats. Meanwhile, Nrf2 and HO-1 expression were upregulated. We demonstrated that inhibition of Nrf-2 expression blocked the inhibitory effect of FXC+UCMSCs-CM on apoptosis and oxidative stress injury. Our data suggest that FXC+UCMSCs may attenuate oxidative stress injury and macroangiopathy in T2DM by activating the Nrf-2/HO-1 pathway.

Abstract 117: AIBP Deficiency Affects Inflammarafts Formation And Macrophage Reprogramming In Atherosclerosis

Background: Excessive accumulation of cholesterol in plasma membrane of vascular macrophages leads to formation of inflammarafts - large, cholesterol-rich lipid rafts hosting inflammatory receptors assemblies - and reprograms macrophages to facilitate hyperinflammatory responses. Removal of cholesterol from membrane and storage in intracellular lipid droplets reduces macrophage inflammatory status. Apolipoprotein A-I binding protein (AIBP) has been found to mediate selective cholesterol efflux and lipid raft dynamics regulation via binding to toll-like receptor-4 (TLR4) thus reducing inflammarafts in activated macrophages. However, the underlying mechanisms driving lipid raft dynamics changes of reprogrammed macrophages remain unclear. The objective of this study was to evaluate the effect of AIBP deficiency on inflammaraft formation and macrophage reprogramming, separately in macrophage foam cells and non-foamy macrophages. Methods: Single-cell suspensions from the aortae of 16-week high-fat diet-fed Apoa1bp -/- Ldlr -/- and Ldlr -/- mice were gated for BODIPY-high foamy and BODIPY-low non-foamy CD45 + F4/80 + macrophages by flow cytometry. Lipid droplets were assessed by BODIPY fluorescence and inflammarafts were characterized by increased levels of lipid rafts and TLR4 dimers. Transcriptome and chromatin accessibility were measured by RNA-seq and ATAC-seq. Plasma lipid levels and size of atherosclerotic lesions and necrotic cores at the aortic origin were measured using standard methods. Results: We found a larger number of foamy macrophages in Apoa1bp -/- Ldlr -/- mice, expressing significantly higher levels of lipid rafts and TLR4 dimers than those in Ldlr -/- mice. However, there were no inflammarafts differences between non-foamy macrophages in Apoa1bp -/- Ldlr -/- and Ldlr -/- mice. The inflammarafts measures were associated with plasma cholesterol levels and size of atherosclerotic lesions and necrotic cores. Inflammatory pathways were up-regulated in Apoa1bp -/- Ldlr -/- foamy macrophages, compared to Ldlr -/- mice. Conclusions: Our results suggest that AIBP helps reduce inflammarafts specifically in macrophage foam cells, rendering them less inflammatory as shown by transcriptomic and open chromatin analyses.

An overview of the cholesterol metabolism and its proinflammatory role in the development of MASLD.

Cholesterol is an essential lipid molecule in mammalian cells. It is not only involved in the formation of cell membranes but also serves as a raw material for the synthesis of bile acids, vitamin D, and steroid hormones. Additionally, it acts as a covalent modifier of proteins and plays a crucial role in numerous life processes. Generally, the metabolic processes of cholesterol absorption, synthesis, conversion, and efflux are strictly regulated. Excessive accumulation of cholesterol in the body is a risk factor for metabolic diseases such as cardiovascular disease, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease (MASLD). In this review, we first provide an overview of the discovery of cholesterol and the fundamental process of cholesterol metabolism. We then summarize the relationship between dietary cholesterol intake and the risk of developing MASLD, and also the animal models of MASLD specifically established with a cholesterol-containing diet. In the end, the role of cholesterol-induced inflammation in the initiation and development of MASLD is discussed.

Tumor-secreted FGF21 acts as an immune suppressor by rewiring cholesterol metabolism of CD8+T cells

Tumors employ diverse strategies for immune evasion. Unraveling the mechanisms by which tumors suppress anti-tumor immunity facilitates the development of immunotherapies. Here, we have identified tumor-secreted fibroblast growth factor 21 (FGF21) as a pivotal immune suppressor. FGF21 is upregulated in multiple types of tumors and promotes tumor progression. Tumor-secreted FGF21 significantly disrupts anti-tumor immunity by rewiring cholesterol metabolism of CD8+Tcells. Mechanistically, FGF21 sustains the hyperactivation of AKT-mTORC1-sterol regulatory-element-binding protein 1 (SREBP1) signal axis in the activated CD8+T cells, resulting in the augment of cholesterol biosynthesis and Tcell exhaustion. FGF21 knockdown or blockade using a neutralizing antibody normalizes AKT-mTORC1 signaling and reduces excessive cholesterol accumulation in CD8+T cells, thus restoring CD8+T cytotoxic function and robustly suppressing tumor growth. Our findings reveal FGF21 as a "secreted immune checkpoint" that hampers anti-tumor immunity, suggesting that inhibiting FGF21 could be a valuable strategy to enhance the cancer immunotherapy efficacy.

The Gut Microbiome Affects Atherosclerosis by Regulating Reverse Cholesterol Transport.

The human system's secret organ, the gut microbiome, has received considerable attention. Emerging research has yielded substantial scientific evidence indicating that changes in gut microbial composition and microbial metabolites may contribute to the development of atherosclerotic cardiovascular disease. The burden of cardiovascular disease on healthcare systems is exacerbated by atherosclerotic cardiovascular disease, which continues to be the leading cause of mortality globally. Reverse cholesterol transport is a powerful protective mechanism that effectively prevents excessive accumulation of cholesterol for atherosclerotic cardiovascular disease. It has been revealed how the gut microbiota modulates reverse cholesterol transport in patients with atherosclerotic risk. In this review, we highlight the complex interactions between microbes, their metabolites, and their potential impacts in reverse cholesterol transport. We also explore the feasibility of modulating gut microbes and metabolites to facilitate reverse cholesterol transport as a novel therapy for atherosclerosis.

Increased cholesterol uptake is associated with the altered gene expression in white adipose tissue of ApoE−/− mice fed a high-fat high-cholesterol diet

Apolipoprotein E knock out (ApoE−/−) mice, the widely used model for atherosclerosis, exhibits anti-obesity characteristics due to the impaired lipoprotein internalization. Since excessive accumulation of triglycerides and cholesterol in white adipose tissue (WAT) is shown to increase the risk of metabolic diseases, we investigated the effects of dietary high-fat high-cholesterol (HFHC) on gene expression profile and the possible role of cholesterol accumulation in WAT of ApoE−/− mice. Control (CON) and HFHC diets were provided to wild-type mice (WC, WH) and ApoE−/− mice (EC, EH) for 10 weeks. Although body and WAT weights were lower in the ApoE−/− group compared to the wild-type group, increases in cholesterol and lipid peroxides in WAT were only observed in the ApoE−/− group. Transcriptome analysis revealed 3660 and 839 differentially expressed genes (DEGs) in the EC/WC and EH/WH comparison, respectively. “Thermogenesis” and “Oxidative phosphorylation” KEGG pathways were found in the EC/WC comparison, but not in the EH/WH comparison. We identified 142 and 2585 DEGs in the WH/WC and EH/EC comparison respectively, indicating a stronger effect of HFHC on WAT of ApoE−/− mice. Gene ontology analysis of DEGs revealed the association of DEGs with “Regulation of inflammatory response” term, in the EH/EC comparison, but not in the WH/WC comparison. Especially, genes encoding scavenger receptors and toll-like receptors were associated with cholesterol and lipid peroxide levels in WAT of ApoE−/− mice, but not in wild-type mice. In conclusion, changes in gene expression profile of WAT were more pronounced in ApoE−/− mice compared to wild-type mice in response to HFHC, and these altered genes were related to inflammatory response. These data suggest that increased cholesterol accumulation in WAT by dietary HFHC may play a pivotal role in the regulation of gene expression in ApoE−/− mice.

Investigation of Potential Drug Targets for Cholesterol Regulation to Treat Alzheimer's Disease.

Despite extensive research and seven approved drugs, the complex interplay of genes, proteins, and pathways in Alzheimer's disease remains a challenge. This implies the intricacies of the mechanism for Alzheimer's disease, which involves the interaction of hundreds of genes, proteins, and pathways. While the major hallmarks of Alzheimer's disease are the accumulation of amyloid plaques and tau protein tangles, excessive accumulation of cholesterol is reportedly correlated with Alzheimer's disease patients. In this work, protein-protein interaction analysis was conducted based upon the genes from a clinical database to identify the top protein targets with most data-indicated involvement in Alzheimer's disease, which include ABCA1, CYP46A1, BACE1, TREM2, GSK3B, and SREBP2. The reactions and pathways associated with these genes were thoroughly studied for their roles in regulating brain cholesterol biosynthesis, amyloid beta accumulation, and tau protein tangle formation. Existing clinical trials for each protein target were also investigated. The research indicated that the inhibition of SREBP2, BACE1, or GSK3B is beneficial to reduce cholesterol and amyloid beta accumulation, while the activation of ABCA1, CYP46A1, or TREM2 has similar effects. In this study, Sterol Regulatory Element-Binding Protein 2 (SREBP2) emerged as the primary protein target. SREBP2 serves a pivotal role in maintaining cholesterol balance, acting as a transcription factor that controls the expression of several enzymes pivotal for cholesterol biosynthesis. Novel studies suggest that SREBP2 performs a multifaceted role in Alzheimer's disease. The hyperactivity of SREBP2 may lead to heightened cholesterol biosynthesis, which suggested association with the pathogenesis of Alzheimer's disease. Lowering SREBP2 levels in an Alzheimer's disease mouse model results in reduced production of amyloid-beta, a major contributor to Alzheimer's disease progression. Moreover, its thoroughly analyzed crystal structure allows for computer-aided screening of potential inhibitors; SREBP2 is thus selected as a prospective drug target. While more protein targets can be added onto the list in the future, this work provides an overview of key proteins involved in the regulation of brain cholesterol biosynthesis that may be further investigated for Alzheimer's disease intervention.

The human microglial surveillant phenotype is preserved by de novo neurosteroidogenesis through the control of cholesterol homeostasis: Crucial role of 18 kDa Translocator Protein

Neurodegenerative disease-associated microglia commonly exhibit harmful cholesterol accumulation that impairs their ability to resolve the neuroinflammatory response, contributing to disease onset and progression. Neurosteroids, whose levels have been often found significantly altered in brain diseases, are the most potent endogenous anti-inflammatory molecules exerting beneficial effects on activities of brain cells, including microglia. For the first time, the impact of neurosteroidogenesis on cholesterol homeostasis for the immune surveillance phenotype maintenance was investigated in a human microglia in vitro model. To enhance and inhibit neurosteroidogenesis, pharmacological stimulation and knock-down of 18 kDa Translocator Protein (TSPO), which is involved in the neurosteroidogenesis rate-limiting step, were used as experimental approaches, respectively. The obtained results point to an essential autocrine control of neurosteroidogenesis in orchestrating cholesterol trafficking in human microglia. TSPO pharmacological stimulation ensured cholesterol turnover by strengthening cholesterol efflux systems and preserving healthy immune surveillant phenotype. Conversely, TSPO knock-down induced an impairment of the controlled interplay among cholesterol synthesis, efflux, and metabolism mechanisms, leading to an excessive cholesterol accumulation and acquisition of a chronically activated dysfunctional phenotype. In this model, the exogenous neurosteroid administration restored proper the cholesterol clearance. The TSPO ability in promoting native neurosteroidogenesis opens the way to restore cholesterol homeostasis, and thus to maintain microglia proper functionality for the treatment of neuroinflammation-related brain diseases.

Abstract 468: Nano-targeting Epsin In Cholesterol Uptake-efflux Fortifies Atheroma Regression

Excess cholesterol accumulation in lesional macrophages elicits complex responses in atherosclerosis. Epsins, endocytic adaptors, fuel the progression of atherosclerosis; however, the underlying mechanism and therapeutic potential of targeting Epsins remains unknown. In our study, we determined the role of Epsins in macrophage-mediated metabolic regulation and developed a method to target macrophage Epsins with specially-designed S2P-conjugated lipid nanoparticles (NPs), which encapsulate siRNAs to suppress Epsins.We used scRNA-seq with our newly developed algorithm MEBOCOST to study cell-cell communications mediated by metabolites from sender cells and sensor proteins on receiver cells. Biomedical, cellular and molecular approaches were utilized to investigate the role of macrophage Epsins in regulating lipid metabolism and transport. The NPs targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis.We revealed that Epsins regulate lipid metabolism and transport in atherosclerotic macrophages. Inhibiting Epsins by nanotherapy halts inflammation and accelerates atheroma resolution. Harnessing macrophage-specific NP delivery of Epsin siRNAs, we showed that silencing of macrophage Epsins markedly diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, we demonstrated that Epsins bound to CD36 to facilitate lipid uptake by enhancing CD36 endocytosis and recycling. Conversely, Epsins promoted ABCG1 degradation via lysosomes and hampered ABCG1-mediated cholesterol efflux and reverse cholesterol transport.Our findings suggest that targeting Epsins in lesional macrophages may offer therapeutic benefits for advanced atherosclerosis.

Resveratrol Modulates the Redox Response and Bile Acid Metabolism to Maintain the Cholesterol Homeostasis in Fish Megalobrama amblycephala Offered a High-Carbohydrate Diet.

This study aimed to characterize the effects of resveratrol on the redox balance, cholesterol homeostasis and bile acid metabolism of Megalobrama amblycephala offered a high-carbohydrate diet. Fish (35.0 ± 0.15 g) were fed four diets including one control diet (32% nitrogen-free extract), one high-carbohydrate diet (45% nitrogen-free extract, HC), and the HC diet supplemented with different levels (0.04%, HCR1; 0.08%, HCR2) of resveratrol for 12 weeks. The HC diet-induced redox imbalance is characterized by increased MDA content and decreased T-SOD and CAT activities in the liver. Resveratrol attenuated this by up-regulating the transcription of Cu/Zn-sod, and increasing the activities of T-SOD, CAT, and GPX. The HC diet enhanced the cholesterol synthesis, but decreased the bile acid synthesis via up-regulating both hmgcr and acat2, and down-regulating cyp7a1, thus resulting in excessive cholesterol accumulation. Resveratrol supplement decreased cholesterol synthesis, and increased cholesterol uptake in the liver by down-regulating both hmgcr and acat2, and up-regulating ldlr. It also increased bile acid synthesis and biliary excretion by up-regulating cyp7a1, and down-regulating mrp2, oatp1, and oatp4 in the hindgut, thereby decreasing cholesterol accumulation. In conclusion, resveratrol improves the cholesterol homeostasis of Megalobrama amblycephala fed a high-carbohydrate diet by modulating the redox response and bile acid metabolism.

Unresolved Excess Accumulation of Myelin-Derived Cholesterol Contributes to Scar Formation after Spinal Cord Injury.

Spinal cord injury triggers complex pathological cascades, resulting in destructive tissue damage and incomplete tissue repair. Scar formation is generally considered a barrier for regeneration in the central nervous system. However, the intrinsic mechanism of scar formation after spinal cord injury has not been fully elucidated. Here, we report that excess cholesterol accumulates in phagocytes and is inefficiently removed from spinal cord lesions in young adult mice. Interestingly, we observed that excessive cholesterol also accumulates in injured peripheral nerves but is subsequently removed by reverse cholesterol transport. Meanwhile, preventing reverse cholesterol transport leads to macrophage accumulation and fibrosis in injured peripheral nerves. Furthermore, the neonatal mouse spinal cord lesions are devoid of myelin-derived lipids and can heal without excess cholesterol accumulation. We found that transplantation of myelin into neonatal lesions disrupts healing with excessive cholesterol accumulation, persistent macrophage activation, and fibrosis. Myelin internalization suppresses macrophage apoptosis mediated by CD5L expression, indicating that myelin-derived cholesterol plays a critical role in impaired wound healing. Taken together, our data suggest that the central nervous system lacks an efficient approach for cholesterol clearance, resulting in excessive accumulation of myelin-derived cholesterol, thereby inducing scar formation after injury.

Epsin Nanotherapy Regulates Cholesterol Transport to Fortify Atheroma Regression.

Excess cholesterol accumulation in lesional macrophages elicits complex responses in atherosclerosis. Epsins, a family of endocytic adaptors, fuel the progression of atherosclerosis; however, the underlying mechanism and therapeutic potential of targeting Epsins remains unknown. In this study, we determined the role of Epsins in macrophage-mediated metabolic regulation. We then developed an innovative method to therapeutically target macrophage Epsins with specially designed S2P-conjugated lipid nanoparticles, which encapsulate small-interfering RNAs to suppress Epsins. We used single-cell RNA sequencing with our newly developed algorithm MEBOCOST (Metabolite-mediated Cell Communication Modeling by Single Cell Transcriptome) to study cell-cell communications mediated by metabolites from sender cells and sensor proteins on receiver cells. Biomedical, cellular, and molecular approaches were utilized to investigate the role of macrophage Epsins in regulating lipid metabolism and transport. We performed this study using myeloid-specific Epsin double knockout (LysM-DKO) mice and mice with a genetic reduction of ABCG1 (ATP-binding cassette subfamily G member 1; LysM-DKO-ABCG1fl/+). The nanoparticles targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis. We revealed that Epsins regulate lipid metabolism and transport in atherosclerotic macrophages. Inhibiting Epsins by nanotherapy halts inflammation and accelerates atheroma resolution. Harnessing lesional macrophage-specific nanoparticle delivery of Epsin small-interfering RNAs, we showed that silencing of macrophage Epsins diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, we demonstrated that Epsins bound to CD36 to facilitate lipid uptake by enhancing CD36 endocytosis and recycling. Conversely, Epsins promoted ABCG1 degradation via lysosomes and hampered ABCG1-mediated cholesterol efflux and reverse cholesterol transport. In a LysM-DKO-ABCG1fl/+ mouse model, enhanced cholesterol efflux and reverse transport due to Epsin deficiency was suppressed by the reduction of ABCG1. Our findings suggest that targeting Epsins in lesional macrophages may offer therapeutic benefits for advanced atherosclerosis by reducing CD36-mediated lipid uptake and increasing ABCG1-mediated cholesterol efflux.

HDLBP Promotes Hepatocellular Carcinoma Proliferation and Sorafenib Resistance by Suppressing Trim71-dependent RAF1 Degradation

The contribution of abnormal metabolic targets to hepatocellular carcinoma (HCC) progression and the associated regulatory mechanisms are attractive research areas. High-density lipoprotein binding protein (HDLBP) is an important transporter that protects cells from excessive cholesterol accumulation, but few studies have identified a role for HDLBP in HCC progression. HDLBP expression was determined in HCC tissues and published datasets. The biological roles of HDLBP invitro and invivo were examined by performing a series of functional experiments. An integrated analysis confirmed that HDLBP expression was significantly elevated in HCC compared with noncancerous liver tissues. The knockdown or overexpression of HDLBP substantially inhibited or enhanced, respectively, HCC proliferation and sorafenib resistance. Subsequently, a mass spectrometry screen identified RAF1 as a potential downstream target of HDLBP. Mechanistically, when RAF1 was stabilized by HDLBP, MEKK1 continuously induced RAF1Ser259-dependent MAPK signaling. Meanwhile, HDLBP interacted with RAF1 by competing with the TRIM71 E3 ligase and inhibited RAF1 degradation through the ubiquitin-proteasome pathway. Our study reveals that HDLBP is an important mediator that stabilizes the RAF1 protein and maintains its activity, leading to HCC progression and sorafenib resistance. Thus, HDLBP might represent a potential biomarker and future therapeutic target for HCC.

Nano-sponge-like liposomes remove cholesterol crystals for antiatherosclerosis.

Atherosclerotic cardiovascular diseases remain the leading causes of morbidity and mortality worldwide. Cholesterol crystals in atherosclerotic plaques play an essential role in atherosclerosis progression. However, no clinical drugs have been used for removing cholesterol crystals from plaque to counter atherosclerosis. Previous studies identified the hydrophobic domain of lipid bilayer in liposomes acted as sinks for solubilizing hydrophobic cholesterol. Moreover, adjusting the composition of the lipid bilayer in liposomes can enhance its hydrophobic molecule loading capacity. Therefore, in this study, ginsenosides Rb1 (Rb1), one of main active components of ginseng which has a similar structure to cholesterol, is anchored into soy phospholipids bilayer with its hydrophobic region to prepare nano-sponge-like liposomes (Rb1-LPs), aiming to amplify the solubilization of cholesterol in lipid bilayer. For targeting delivery to atherosclerotic plaques, Annexin V (AnxV), a protein that can specifically recognize phosphatidylserine upregulated in atherosclerotic plaques, is applied to decorate the surface of Rb1-LPs by click reaction to obtain the final preparation of AnxV-Rb1-LPs. The in vitro studies showed that incorporating Rb1 into lipid bilayer remarkably increased the affinity of the lipid bilayer to free cholesterol and the solubilization of cholesterol crystals. Additionally, nano-sponge-like liposomes could efficiently reduce the accumulation of cholesterol crystals and improve cholesterol efflux, finally inhibiting inflammation and apoptosis in cholesterol-laden cells. Furthermore, AnxV-Rb1-LPs could efficiently accumulate in atherosclerotic plaques after intravenous injection, exert nano-sponge-like functions to remove intra- and extracellular cholesterol crystals, ultimately alleviating inflammation and apoptosis in atherosclerotic plaques for antiatherosclerosis. Therefore, AnxV-Rb1-LPs provide a potential strategy for removing cholesterol crystals in atherosclerotic plaques and can be further utilized in other diseases with excessive cholesterol accumulation.

LIMP2 gene, evolutionarily conserved regulation by TFE3, relieves lysosomal stress induced by cholesterol

AimExcess cholesterol deposition in lysosomes may result in lysosomal stress and dysfunction. Here, we focus on the role of lysosome membrane protein 2 (LIMP2) in relieving the lysosomal stress caused by excess cholesterol and the mechanism that regulate its expression. Material and methodsCholesterol enrichment in lamprey liver tissue was evaluated by RNA transcriptome data analysis, RT-qPCR, H&E, and Oil Red O staining. Gene markers of autophagy and cholesterol synthesis were determined by western blot or RT-qPCR. Lysosomal morphology and pH value was measured by confocal observation or flow cytometry. Dual-Luciferase reporter assay was performed to test the expression regulation relationship. Key findingsWe report that lamprey limp2 (L-limp2) is evolutionarily highly conserved with human LIMP2 (H-LIMP2). The biological function of L-limp2, consistent with H-LIMP2, includes maintaining lysosomal morphology, modulating autophagy, and aiding cholesterol efflux from lysosomes. Furthermore, we find that both L-limp2 and H-limp2 can restore cholesterol-induced elevation of lysosomal pH and impaired autophagic flux. We demonstrate that lamprey transcription factor binding to IGHM enhancer 3 (L-TFE3) can bind with coordinated lysosomal expression and regulation (CLEAR) elements on the L-limp2 promoter and regulate its expression. Moreover, this regulatory relationship is also available in humans. Taken together, the present study demonstrates that the evolutionarily conserved TFE3-LIMP2 axis may have a protective role against the impaired lysosomal function caused by excess cholesterol. SignificanceThe protective effect of TFE3-LIMP2 axis against cholesterol-triggered lysosomal stress may provide a new target for the treatment of diseases caused by excessive cholesterol accumulation in lysosomes.

Abstract 506: Targeting Macrophage Epsins Inhibits Lipid Uptake And Facilitates Cholesterol Efflux In Atherosclerosis

Resolving atheroma and hindering their transition to vulnerable atherosclerotic plaque is pivotal in preventing deadly episodes such as heart attacks and strokes. Despite recent advances in the treatment, atherosclerosis remains the root cause of death worldwide. Excess cholesterol accumulation and deposition trigger a complex inflammatory response in atherosclerosis. Our recent studies demonstrate that epsins, a family of endocytic adaptors, are critically responsible for the aforesaid events in atherosclerosis; however, the underlying mechanisms and whether targeting epsins hinders cholesterol uptake, promote cholesterol efflux and reverse transport in macrophages to reduce inflammation and accelerate atheroma resolution remains an open question. To explore the therapeutic intervention by harnessing recent development in nanomedicine, we silenced epsins in lesional macrophages by specific delivery of nanoparticles (NPs) carrying epsin siRNAs. We showed that inhibit epsins significantly diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, epsins bind to CD36 to facilitate CD36-mediated lipid uptake by enhancing its endocytosis and recycling. Conversely, epsins promote ABCG1 degradation via lysosomes and hamper ABCG1-mediated cholesterol efflux and reverse cholesterol transport. Furthermore, myeloid-specific epsins double knockout mice (LysM-DKO) harboring a genetically reduced ABCG1 level (LysM-DKO-ABCG1 fl/+ ) were generated. In LysM-DKO-ABCG1 fl/+ macrophages, enhanced cholesterol efflux and reverse transport due to epsins deficiency was suppressed. Our findings suggest that targeting epsins in lesional macrophages may offer therapeutic benefits in preventing or treating advanced atherosclerosis.

Abstract 207: Autophagy Is Differentially Regulated In Leukocyte And Nonleukocyte Foam Cells During Atherosclerosis

Atherosclerosis is characterized by a build-up of foam cells in the arterial wall, resulting from excess cholesterol uptake and accumulation of cytosolic lipid droplets (LDs). Autophagy has been shown to be atheroprotective in part by promoting the catabolism of LDs which liberates free cholesterol for efflux out of foam cells to cholesterol acceptors (ApoA-I or HDL) for removal from the body. Apart from macrophages (MΦ), vascular smooth muscle cells (VSMCs) comprise 50-70% of foam cells in the plaques. Unlike MΦ, the capacity of VSMC foam cells to metabolize cholesterol via autophagy is unknown. Here, we performed a comparative analysis of the autophagic capacity and cholesterol efflux of arterial foam cell subtypes of the atherosclerotic plaque. Atherosclerosis was induced in hypercholesterolemic autophagy reporter mice (GFP-LC3 mice receiving PCSK9-AAV and fed a Western diet). Autophagic flux in aortic digests was assessed by quantifying GFP-LC3 fluorescence after ex vivo treatment with the autophagy inhibitor Bafilomycin A1 or vehicle. MΦ foam cells displayed functional autophagy as shown by an accumulation of GFP-LC3 upon autophagy inhibition. In contrast, VSMC foam cells did not similarly accumulate GFP-LC3 upon bafilomycin treatment, suggesting dysfunction autophagy in these cells. Additionally, immunostaining of late-stage aortic roots showed MΦ, but not VSMC, foam cells induction of the active autophagy marker pATG16L1. Cell culture studies of lipid loaded MΦ and VSMC corroborated this inability for VSMCs to initiate autophagy in vivo . In vitro , MΦ foam cells effluxed cholesterol to ApoA-I (14%) and HDL (50%), whereas VSMC foam cells minimally effluxed cholesterol to HDL (7%) but not apoA-I. However, unlike MΦ foam cells, VSMC efflux was pharmacologically induced by treatment with metformin. Our data therefore demonstrates a lack of functional autophagy in VSMC, as compared to MΦ foam cells, which impairs their ability to perform cholesterol efflux. This autophagy defect in VSMC foam cells can be increased by autophagy activation using metformin, highlighting both the importance of understanding cholesterol metabolism in all foam cell populations and a new avenue to treat atherosclerosis.