Promote Cholesterol Efflux Research Articles

Abstract 4119262: Anti-atherosclerotic Potential of M2 Macrophage-Derived Exosomes by Promoting Cholesterol Efflux of Foam Cell-Like Vascular Smooth Muscle Cells via the PPARγ-LXRα-ABCA1/ ABCG1 Pathway

Background: Impediment in the excretion of lipid deposits within SMC-derived foam cells (SMC-FCs) is one of the reasons for the continuous expansion of the plaque necrotic core. This study aims to explore the effect and underlying mechanimsm of exosomes secreted by M2 macrophage (M2-exos) on SMC-FCs lipid metabolism and plaque stability. Methods: First, immunofluorescence was used to detect the expression levels of CD45 (a recognized differentially expressed molecule of myeloid and SMC-FCs) and the key proteins of cholesterol efflux pathway, ABCA1 and ABCG1, in human early and late plaques. Exosomes derived from M0 and M2 macrophages were purified by sucrose density gradient centrifugation, and characterized based on specific morphology and surface markers. Western blot, Oil red O staining and cell total cholesterol assay were used to assess the effects of M2-exos on the lipid mechanism of SMC-FCs. RNA-seq was used to detect the miRNA profiles of M2-exos. Quantitative real-time PCR was used to identify candidate miRNAs. The dual-luciferase reporting system was utilized to assess the regulatory effect of candidate miRNA on target gene. Then, the effect of M2-exos on the progression and stability of plaques in ApoE -/- mice was evaluated using Oil red O, H&E, Masson, Movat and immunohistochemistry. Results: Immunofluorescence revealed that compared with early plaques, VSMC-FCs (CD45 - ) were significantly increased in late plaques, and the expression levels of ABCG1 and ABCA1 were marked lower than those in macrophage-derived foam cells (CD45 + ). Purified M2-exos treatment significantly promoted the cholesterol efflux of SMC-FCs in vitro. In high-fat-fed ApoE -/- mice, M2-exos significantly reduced the VSMC-FCs, delayed the progression of plaques, decreased necrotic core area and enhanced plaque stability. MiRNA profiling and comprehensive analysis of signaling pathways showed that M2-exos were rich in miR-7683-3p, which played a key role in regulating SMC-FCs lipid metabolism through PPARγ-LXRα-ABCA1/ABCG1 pathway. Dual-luciferase reporting assay showed that miR-7683-3p can specifically bind to the promoter region of homeobox genes A1(HoxA1), an inhibitor of PPARγ-LXRα-ABCA1/ABCG1 pathway. Conclusion: M2-exos exerted an obvious atherosclerotic protective effect, and the underlying mechanism was closely related to MiR-7683-3p, which targeted the 3’UTR of HoxA1 mRNA and activated the PPARγ-LXRα-ABCA1/ABCG1 mediated cholesterol efflux in SMC-FCs.

Docosahexaenoic acid alleviated liver lipid deposition and health damage induced by cholesterol accumulation in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂)

Cholesterol accumulation can be a critical pathological marker and, while, the cholesterol-lowering potential of docosahexaenoic acid (DHA) has been documented extensively in mammals, its effects in fish are unclear. The present study investigated the adverse effects of cholesterol accumulation on liver health, and evaluated DHA as a potential cholesterol-lowering agent to mitigate liver damage induced by high cholesterol (HC) intake in hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂). Grouper were fed either a control diet or an HC (1.6 %) diet supplemented with varying levels of DHA (0, 0.5, 1, 2 %) for eight weeks. The HC diet significantly increased condition factor (CF), mesenteric fat index (MFI), and lipid, cholesterol and triglyceride accumulation in liver. Supplementing the HC diet with 0.5 % DHA significantly reduced CF, MFI and liver lipid contents. Moreover, HC intake increased aspartate transaminase (AST) and alanine transaminase (ALT) activities in serum, and malondialdehyde (MDA) content and the expression of gene related to inflammatory responses and apoptosis in liver. In addition, HC intake significantly reduced mitochondrial biogenesis and total antioxidant capacity in liver. However, supplementing the HC diet with 0.5 % DHA significantly decreased liver cholesterol accumulation by inhibiting cholesterol synthesis and promoting cholesterol efflux, and significantly reduced liver triglyceride content by inhibiting the expression of genes associated with lipogenesis and lipid droplet formation. Furthermore, supplementation of the HC diet with 0.5 % DHA significantly reduced the activities of ALT and AST in serum, and MDA content and the expression of genes associated with inflammation, apoptosis and antioxidation in liver, and enhanced liver mitochondrial biogenesis. Overall, the results indicated that DHA supplementation mitigated liver cholesterol accumulation by inhibiting cholesterol synthesis and promoting cholesterol efflux, and enhanced mitochondrial function, boosted antioxidant capacity, and reduced inflammation in liver of grouper fed the HC diet, ultimately alleviating liver damage caused by cholesterol accumulation. The present study elucidated the detrimental effects of cholesterol accumulation on liver health in aquatic animals and proposed DHA as a potential intervention for mitigating health issues associated with cholesterol overload.

Biomimetic nanoparticles co-deliver hirudin and lumbrukinase to ameliorate thrombus and inflammation for atherosclerosis therapy

Atherosclerosis (AS) is a progressive inflammatory disease, and thrombosis most probably leads to cardiovascular morbidity and mortality globally. Thrombolytic drugs alone cannot completely prevent thrombotic events, and treatments targeting thrombosis also need to regulate the inflammatory process. Based on the pathological process of AS dynamic development, biomimetic thrombus-targeted nanoparticles HMTL@PM were prepared. Hirudin and lumbrukinase, the effective substances of traditional Chinese medicine, were self-assembled under the action of tannic acid and Mn2+. HMTL@PM dissociated in the weakly acidic microenvironment of atherosclerosis and exhibited an excellent therapeutic effect of alleviating inflammation, dissolving thrombus, anticoagulation, and promoting cholesterol efflux. HMTL@PM effectively regulated the progression of AS and provided a new perspective for the development of drug delivery systems for AS therapy, which owned important research significance for reducing the mortality of cardiovascular and cerebrovascular diseases.

Apolipoprotein A1-encoding recombinant adenovirus remodels cholesterol metabolism in tumors and the tumor microenvironment to inhibit hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a prevalent malignant tumor requiring effective treatments. Oncolytic viruses induce anti-tumor responses but have limited efficacy. Apolipoprotein A1 (ApoA1) inhibits inflammation, modulates immunity, and promotes anti-oxidation. This study aims to construct an oncolytic adenovirus (Ad5)-ApoA1 for superior anti-tumor effects.We analyzed ApoA1 expression in tumors and its prognostic significance using public databases. Subsequently, we engineered a recombinant oncolytic adenovirus Ad5-ApoA1 and assessed its replication and oncolytic efficacy in vitro and in nude mice. The impact of Ad5-ApoA1 on the tumor microenvironment of HCC was evaluated through flow cytometry, transcriptome sequencing, single-cell sequencing, and other methodologies. Additionally, mechanisms of immune microenvironment modulation by Ad5-ApoA1 were explored. ApoA1 expression was down-regulated with HCC progression and significantly positively correlated with the prognosis of HCC patients. Ad5-ApoA1 exhibited robust oncolytic activity but showed no therapeutic effect on nude mice. However, it significantly inhibited HCC growth and prolonged the survival period of both healthy-immune and humanized immune-reconstituted NCG mice. Furthermore, Ad5-ApoA1 significantly promoted the expression of IFN-γ and GzmB in CD8+ T cells while inhibiting the expression of PD-1 and LAG-3. Notably, the cholesterol content in the CD8+ T cells studied was significantly correlated with the expression of PD-1 and LAG-3, with ApoA1 promoting cholesterol efflux and reducing cholesterol levels. Ad5-ApoA1 activates CD8+ T cells by promoting large-scale viral replication. High levels of ApoA1 protein expression promote cholesterol efflux, inhibit CD8+ T cell depletion, and reduce inflammatory factors, ultimately leading to superior therapeutic effects on hepatocellular carcinoma.

Macrophage membrane-functionalized manganese dioxide nanomedicine for synergistic treatment of atherosclerosis by mitigating inflammatory storms and promoting cholesterol efflux

Atherosclerosis (AS) poses a significant threat to human life and health. However, conventional antiatherogenic medications exhibit insufficient targeting precision and restricted therapeutic effectiveness. Moreover, during the progression of AS, macrophages undergo polarization toward the proinflammatory M1 phenotype and generate reactive oxygen species (ROS) to accelerate the occurrence of inflammatory storms, and ingest excess lipids to form foam cells by inhibiting cholesterol efflux. In our study, we developed a macrophage membrane-functionalized hollow mesoporous manganese dioxide nanomedicine (Col@HMnO2-MM). This nanomedicine has the ability to evade immune cell phagocytosis, enables prolonged circulation within the body, targets the inflammatory site of AS for effective drug release, and alleviates the inflammatory storm at the AS site by eliminating ROS. Furthermore, Col@HMnO2-MM has the ability to generate oxygen autonomously by breaking down surplus hydrogen peroxide generated at the inflammatory AS site, thereby reducing the hypoxic microenvironment of the plaque by downregulating hypoxia-inducible factor (HIF-1α), which in turn enhances cholesterol efflux to inhibit foam cell formation. In an APOE−/− mouse model, Col@HMnO2-MM significantly reduced inflammatory factor levels, lipid storage, and plaque formation without significant long-term toxicity. In summary, this synergistic treatment significantly improved the effectiveness of nanomedicine and may offer a novel strategy for precise AS therapy.

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

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

Blue Mussel-Derived Bioactive Peptides PIISVYWK (P1) and FSVVPSPK (P2): Promising Agents for Inhibiting Foam Cell Formation and Inflammation in Cardiovascular Diseases.

Atherosclerosis is a key etiological event in the development of cardiovascular diseases (CVDs), strongly linked to the formation of foam cells. This study explored the effects of two blue mussel-derived bioactive peptides (BAPs), PIISVYWK (P1) and FSVVPSPK (P2), on inhibiting foam cell formation and mitigating inflammation in oxLDL-treated RAW264.7 macrophages. Both peptides significantly suppressed intracellular lipid accumulation and cholesterol levels while promoting cholesterol efflux by downregulating cluster of differentiation 36 (CD36) and class A1 scavenger receptors (SR-A1) and upregulating ATP binding cassette subfamily A member 1 (ABCA-1) and ATP binding cassette subfamily G member 1 (ABCG-1) expressions. The increased expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α) further validated their role in enhancing cholesterol efflux. Additionally, P1 and P2 inhibited foam cell formation in oxLDL-treated human aortic smooth muscle cells and exerted anti-inflammatory effects by reducing pro-inflammatory cytokines, nitric oxide (NO), prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), primarily through inhibiting NF-κB activation. Furthermore, P1 and P2 alleviated oxidative stress by activating the Nrf2/HO-1 pathway. Our findings demonstrate that P1 and P2 have significant potential in reducing foam cell formation and inflammation, both critical factors in atherosclerosis development. These peptides may serve as promising therapeutic agents for the prevention and treatment of CVDs associated with oxidative stress and inflammation.

Cholesterol Efflux Decreases TLR4-Target Gene Expression in Cultured Macrophages Exposed to T. brucei Ghosts.

Trypanosoma brucei causes African trypanosomiasis in humans. Infection with T. brucei elicits a potent pro-inflammatory immune response within infected human hosts, and this response is thought to at least be partially due to Toll-like receptor (TLR) activation. In response to stimulation by lipopolysaccharide and other pathogen antigens, TLR4 translocates to lipid rafts, which induces the expression of pro-inflammatory genes. However, cholesterol efflux is acknowledged as anti-inflammatory due to promoting lipid raft disruption. In this study, we wanted to assess the impact of T. brucei "ghosts", which are non-viable T. brucei essentially devoid of intracellular contents, in stimulating macrophage TLR4 translocation to lipid rafts, and whether promoting cholesterol efflux in macrophages incubated with T. brucei ghosts attenuates TLR4-target gene expression. When cultured macrophages were exposed to T. brucei ghosts, we observed an increase in lipid raft TLR4 protein content, which suggests certain surface molecules of T. brucei serve as ligands for TLR4. However, pretreating macrophages with cholesterol acceptors before T. brucei ghost exposure decreased lipid raft TLR4 protein content and the expression of pro-inflammatory TLR4-target genes. Taken together, these results imply that macrophage cholesterol efflux weakens pro-inflammatory responses which occur from T. brucei infection via increasing macrophage lipid raft disruption.

The Lian-Dou-Qing-Mai Formula activates the PPARγ-LXRα-ABCA1/ABCG1 pathway by regulating IL-10, leading to the promotion of cholesterol efflux and a reduction in atherosclerotic plaques.

To observe the effect of the Lian-Dou-Qing-Mai (LDQM) formula on lipid metabolism in mice and explore its mechanism from the perspective of regulating the PPARγ/LXRα/ABCA1 signaling pathway. THP-1 cells were induced to transform into foam cells with ox-LDL. Atherosclerosis (AS) models were constructed using a high-fat diet in ApoE-/- mice. Detection kits were used to evaluate triglyceride (TG) and total cholesterol (TC) content; TNF-α, MCP-1, MMP-9, TMP-1, PPARγ, LXRα, ABCA1, and ABCG1 mRNA and protein expression were identified using real-time PCR and western blot. Interleukin-10 (IL-10) blood levels were measured using an enzyme-linked immunosorbent assay (ELISA), and aortic plaque development and lipid deposition were seen using hematoxylin and eosin (HE) and oil red O staining, respectively. In the cell model, LDQM could inhibit the formation of THP-1 macrophage-derived foam cells and the expression of inflammatory factors, promote macrophage cholesterol efflux, increase the expression of IL-10, and activate the PPARγ-LXRα-ABCA1/ABCG1 pathway. Additional IL-10 treatment further promotes LDQM-induced cholesterol efflux in THP-1 cells; in In vivo models, LDQM inhibited the area of atherosclerotic lesions, aortic lipid deposition, and inflammation levels in ApoE-/- mice through IL-10, and activated the expression level of the PPARγ-LXRα-ABCA1/ABCG1 pathway. LDQM may affect the PPARγ/LXRα/ABCA1 signaling pathway through IL-10, regulate lipid metabolism, reduce serum inflammatory expression and lipid deposition, and improve the formation of atheroplaques.

Cannabidiol (CBD) Inhibits Foam Cell Formation via Regulating Cholesterol Homeostasis and Lipid Metabolism.

The cannabidiol (CBD) in hemp oil has important pharmacological activities. Accumulating evidence suggests that CBD is beneficial in the cardiovascular system and has been applied as a health supplement for atherosclerosis. However, the mechanism remains unclear. This study investigates the impact of CBD on foam cell formation, cholesterol homeostasis, and lipid metabolism in macrophages. CBD elevates the levels of peroxisome proliferator-activated receptor gamma (PPARγ) and its associated targets, such as ATP binding transporter A1/G1 (ABCA1/ABCG1), thus reducing foam cell formation, and increasing cholesterol efflux within macrophages. Notably, the upregulation of ABCA1 and ABCG1 expression induced by CBD is found to be attenuated by both a PPARγ inhibitor and PPARγ small interfering RNA (siRNA). Moreover, transfection of PPARγ siRNA results in a decrease in the inhibitory effect of CBD on foam cell formation and promotion of cholesterol efflux. Through lipidomics analysis, the study finds that CBD significantly reverses the enhancement of ceramide (Cer). Correlation analysis indicates a negative association between Cer level and the expression of ABCA1/ABCG1. This study confirms that CBD can be an effective therapeutic candidate for atherosclerosis treatment by activating PPARγ, up-regulating ABCA1/ABCG1 expression, and down-regulating Cer level.

Ilexgenin A inhibits lipid accumulation in macrophages and reduces the progression of atherosclerosis through PTPN2/ERK1/2/ABCA1 signalling pathway

Macrophage lipid accumulation indicates a pathological change in atherosclerosis. Ilexgenin A (IA), a pentacyclic triterpenoid compound, plays a role in preventing inflammation, bacterial infection, and fatty liver and induces a potential anti-atherogenic effect. However, the anti-atherosclerotic mechanism remains unclear. The present study investigated the effects of IA on lipid accumulation in macrophage-derived foam cells and atherogenesis in apoE−/− mice. Our results indicated that the expression of adenosine triphosphate-binding cassette transporter A1 (ABCA1) was up-regulated by IA, promoting cholesterol efflux and reducing lipid accumulation in macrophages, which may be regulated by the protein tyrosine phosphatase non-receptor type 2 (PTPN2)/ERK1/2 signalling pathway. IA attenuated the progression of atherosclerosis in high-fat diet-fed apoE−/− mice. PTPN2 knockdown with siRNA or treatment with an ERK1/2 agonist (Ro 67–7476) impeded the effects of IA on ABCA1 upregulation and cholesterol efflux in macrophages. These results suggest that IA inhibits macrophage lipid accumulation and alleviates atherosclerosis progression via the PTPN2/ERK1/2 signalling pathway.

Qinghao-Biejia Herb Pair attenuates SLE atherosclerosis by regulating macrophage polarization via ABCA1/G1-mediated cholesterol efflux

Ethnopharmacological relevanceQinghao-Biejia herb pair (QB) is the core herb pair of “Jieduquyuziyin prescription" and is one of the commonly used herb pairs for the clinical treatment of systemic lupus erythematosus (SLE). Previous studies have shown that QB reduces the expression of inflammatory cytokines like IL-6 and TNF-α in the serum and kidney of MRL/lpr mice. Additionally, it inhibits the expression of TLR4 and MyD88 in the kidney and aorta and reduces the deposition of renal complement C3 and aortic plaque after treatment. These findings suggest that QB has a preventive and therapeutic effect on lupus rats. Aim of the studyThis study sought to investigate the mechanisms underlying the anti-SLE combined with atherosclerosis activity of the Qinghao-Biejia herb pair. Materials and methodsDrug targets for QB were identified using the HERB database, while targets associated with SLE and atherosclerosis were retrieved from the GeneCards database. The intersection of these drug and disease targets was then analyzed using a protein-protein interaction (PPI) network with GO and KEGG pathway enrichment analysis. In vivo, apolipoprotein E-deficient (ApoE−/−) mice were induced to develop SLE-AS by intraperitoneal injection of pristane and continued feeding of a high-fat diet. The changes in relevant indexes were observed after 12 weeks of gavage treatment with hydroxychloroquine, QB, Q (Qinghao alone), and B (Biejia alone). Bone marrow-derived macrophages from ApoE−/− mice and Raw 264.7 macrophages were used to explore the mechanisms of QB treatment. ResultsThe levels of inflammatory cytokines in serum and pathological liver changes in mice were improved to varying degrees in the treatment groups. Additionally, there was a reduction in aortic atheromatous plaque formation and some improvement in cholesterol efflux. Furthermore, QB suppressed the expression of inflammatory cytokines in M1 macrophages, suggesting a role in regulating macrophage polarization. ConclusionQB demonstrates clear efficacy for treating SLE-AS, and its therapeutic mechanism may involve the regulation of macrophage phenotypes by promoting cholesterol efflux.

A ROS-responsive multifunctional targeted prodrug micelle for atherosclerosis treatment

Atherosclerosis is a chronic multifactorial cardiovascular disease. To combat atherosclerosis effectively, it is necessary to develop precision and targeted therapy in the early stages of plaque formation. In this study, a simvastatin (SV)-containing prodrug micelle SPCPV was developed by incorporating a peroxalate ester bond (PO). SPCPV could specifically target VCAM-1 overexpressed at atherosclerotic lesions. SPCPV contains a carrier (CP) composed of cyclodextrin (CD) and polyethylene glycol (PEG). At the lesions, CP and SV exerted multifaceted anti-atherosclerotic effects. In vitro studies demonstrated that intracellular reactive oxygen species (ROS) could induce the release of SV from SPCPV. The uptake of SPCPV was higher in inflammatory cells than in normal cells. Furthermore, in vitro experiments showed that SPCPV effectively reduced ROS levels, possessed anti-inflammatory properties, inhibited foam cell formation, and promoted cholesterol efflux. In vivo studies using atherosclerotic rats showed that SPCPV reduced the thickness of the vascular wall and low-density lipoprotein (LDL). This study developed a drug delivery strategy that could target atherosclerotic plaques and treat atherosclerosis by integrating the carrier with SV. The findings demonstrated that SPCPV possessed high stability and safety and had great therapeutic potential for treating early-stage atherosclerosis.

Vitamin D and Atherosclerosis: Unraveling the Impact on Macrophage Function.

Vitamin D plays a crucial role in preventing atherosclerosis and in the regulation of macrophage function. This review aims to provide a comprehensive summary of the clinical evidence regarding the impact of vitamin D on atherosclerotic cardiovascular disease, atherosclerotic cerebrovascular disease, peripheral arterial disease, and associated risk factors. Additionally, it explores the mechanistic studies investigating the influence of vitamin D on macrophage function in atherosclerosis. Numerous findings indicate that vitamin D inhibits monocyte or macrophage recruitment, macrophage cholesterol uptake, and esterification. Moreover, it induces autophagy of lipid droplets in macrophages, promotes cholesterol efflux from macrophages, and regulates macrophage polarization. This review particularly focuses on analyzing the molecular mechanisms and signaling pathways through which vitamin D modulates macrophage function in atherosclerosis. It claims that vitamin D has a direct inhibitory effect on the formation, adhesion, and migration of lipid-loaded monocytes, thus exerting anti-atherosclerotic effects. Therefore, this review emphasizes the crucial role of vitamin D in regulating macrophage function and preventing the development of atherosclerosis.

Untangling the inflammatory responses of liver x receptor (LXR) activation in human macrophages

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Rembrandt Institute for Cardiovascular Science (grant 2022) The regulation of cholesterol and fatty acid homeostasis is essential and the Liver X Receptor (LXR) plays a crucial role in this process. When activated, LXR promotes cholesterol efflux and suppresses the transcription of pro-inflammatory genes in mouse macrophages, making it a promising therapy against atherosclerosis. Studies indeed have shown that LXR agonism is effective in pre-clinical atherosclerotic mouse models. However, the limited data on human macrophages suggests that LXR activation may have additional pro-inflammatory effects in humans, and the mechanisms contributing to this are not yet fully understood. To investigate the inflammatory properties of LXR activation in human macrophages, monocytes were isolated from buffy coats of healthy donors and differentiated into macrophages using M-CSF. The macrophages were exposed to synthetic and endogenous LXR ligands, such as GW3965, T0901317, and desmosterol, respectively, followed by stimulation with lipopolysaccharide (LPS) for six hours. We used qPCR and ELISA to analyze experiments and plan to supplement our data with RNA-Seq and multiplex assays. Preliminary results show that LXR activation reduces LPS-induced activation of interferon-mediated genes like CXCL9 and CXCL10, similar to mice. However, it promotes the transcription of NF-kB target genes like IL1B and IL12B upon LPS activation. These findings suggest that LXR activation in human macrophages not solely suppresses inflammatory responses in macrophages and that LXR activation has different effects on the regulation of genes mediated by interferon or NF-kB. Our research aims to further uncover the regulatory mechanisms behind LXR activation in human macrophages as central regulators in atherosclerosis.

Abstract 2083: Apolipoprotein A-II Increases Cholesterol Efflux Capacity Of HDL Via The Apolipoprotein A-I C-terminus

The ability of high-density lipoprotein (HDL) to promote cellular cholesterol efflux (CEC) is a more robust predictor of cardiovascular disease protection than its plasma quantity. Previously, we found that fully lipidated HDL containing apolipoprotein A-II (APOA2) promotes cholesterol efflux via the ATP binding cassette transporter (ABCA1). This was surprising given that ABCA1 is thought to primarily interact with lipid-poor apolipoproteins. Having previously focused on isolated lipoproteins, we moved into human plasma with the hypothesis APOA2 can enhance ABCA1-mediated CEC in this more complex environment. Human plasma was incubated with increasing amounts of lipid-free APOA2. The samples were assayed for CEC from macrophages +/- cAMP for ABCA1 stimulation. APOA2 dose dependently increased whole plasma CEC - due mostly to HDL as the effect persisted in apoB-depleted plasma. Next, plasma was incubated with lipid-free APOA1 or APOA2, immediately fractionated by size exclusion chromatography and each fraction assayed for CEC. In unmodified plasma, cholesterol effluxed to peaks corresponding to LDL, HDL and lipid-free apolipoproteins with the latter exclusively increased by ABCA1 expression, as expected. When lipid-free APOA1 was added, efflux to LDL and HDL remained largely unchanged. Strikingly, when APOA2 was added, APOA2 increased ABCA1-mediated cholesterol efflux to the fully lipidated HDL fractions. Proteomic analyses indicated that APOA2 increased in HDL though some APOA1 was retained. To explore the mechanism, we reconstituted HDL particles with APOA1 that either contained or lacked its C-terminal lipid binding helix. APOA2 lost the ability to stimulate ABCA1 efflux to HDL when the C-terminal domain of APOA1 was deleted. Our current hypothesis is that APOA2 displaces the C-terminal helix of APOA1 from the HDL surface which can then interact with ABCA1 - much like it does in lipid-poor APOA1. Reconstituted forms of APOA1 are under development as a therapy to rapidly clear cholesterol from coronary arteries in patients with acute coronary syndrome. Our work suggests APOA2 may be a novel target given its dual benefits for cholesterol efflux.

Abstract 3039: TOMM40 Knockdown In Macrophages Inhibits Oxidized LDL-induced NLRP3 Activation And Promotes Reverse Cholesterol Transport

Atherogenesis has been shown to be promoted by activation of the NLRP3 inflammasome, a cytosolic innate immune sensor activated by a broad range of factors, including oxidized LDL (oxLDL). Our group has shown that hepatocyte knockdown of Translocase of Outer Mitochondrial Membrane 40 ( TOMM40), which encodes a key component of the mitochondrial importer TOM complex, results in increased levels of oxysterols that activate the transcription factors LXRα and β. As LXR agonists have been shown to inhibit NLRP3 activation, we tested whether TOMM40 knockdown reduces NLRP3 activation in THP-1 monocyte-derived macrophages. On stimulating these cells with both the NLRP3 activator Nigericin and oxLDL following TOMM40 knockdown by siRNA, we found that pro-inflammatory cytokine release and NLRP3 activation were significantly inhibited (Two-way ANOVA, p<0.0001, n=6). We further showed that TOMM40 knockdown resulted in significant LXRβ upregulation (unpaired t-test, p=0.007, n=3), and reduced oxLDL-induced upregulation of both NLRP3 and CASP1 , which encodes a protein recruited in NLRP3 activation (One-way ANOVA, p=0.0007, n=3). Moreover, pre-treatment with an LXR antagonist, GSK2033, abrogated TOMM40 knockdown-induced NLRP3 inhibition, supporting our hypothesis that this inhibition is likely LXR dependent. Since LXRs are known to promote cholesterol efflux from macrophages, we also examined the effect of TOMM40 knockdown on expression of the cholesterol transporter genes, ABCA1, ABCG1, and SR-B1 , and found that ABCG1 and SR-B1 were significantly upregulated (unpaired t-test, p=0.02 and p=0.003 respectively, n=3). Taken together, these findings identify mechanisms by which TOMM40 knockdown exerts anti-inflammatory effects and promotes reverse cholesterol transport. While TOMM40 has been studied largely in relation to APOE and neurodegenerative disease due to its genomic location, we identify a novel potential impact of TOMM40 on atherogenesis.

Oncolytic adenovirus encoding apolipoprotein A1 suppresses metastasis of triple-negative breast cancer in mice

BackgroundDysregulation of cholesterol metabolism is associated with the metastasis of triple-negative breast cancer (TNBC). Apolipoprotein A1 (ApoA1) is widely recognized for its pivotal role in regulating cholesterol efflux and maintaining cellular cholesterol homeostasis. However, further exploration is needed to determine whether it inhibits TNBC metastasis by affecting cholesterol metabolism. Additionally, it is necessary to investigate whether ApoA1-based oncolytic virus therapy can be used to treat TNBC.MethodsIn vitro experiments and mouse breast cancer models were utilized to evaluate the molecular mechanism of ApoA1 in regulating cholesterol efflux and inhibiting breast cancer progression and metastasis. The gene encoding ApoA1 was inserted into the adenovirus genome to construct a recombinant adenovirus (ADV-ApoA1). Subsequently, the efficacy of ADV-ApoA1 in inhibiting the growth and metastasis of TNBC was evaluated in several mouse models, including orthotopic breast cancer, spontaneous breast cancer, and human xenografts. In addition, a comprehensive safety assessment of Syrian hamsters and rhesus monkeys injected with oncolytic adenovirus was conducted.ResultsThis study found that dysregulation of cholesterol homeostasis is critical for the progression and metastasis of TNBC. In a mouse orthotopic model of TNBC, a high-cholesterol diet promoted lung and liver metastasis, which was associated with keratin 14 (KRT14), a protein responsible for TNBC metastasis. Furthermore, studies have shown that ApoA1, a cholesterol reverse transporter, inhibits TNBC metastasis by regulating the cholesterol/IKBKB/FOXO3a/KRT14 axis. Moreover, ADV-ApoA1 was found to promote cholesterol efflux, inhibit tumor growth, reduce lung metastasis, and prolonged the survival of mice with TNBC. Importantly, high doses of ADV-ApoA1 administered intravenously and subcutaneously were well tolerated in rhesus monkeys and Syrian hamsters.ConclusionsThis study provides a promising oncolytic virus treatment strategy for TNBC based on targeting dysregulated cholesterol metabolism. It also establishes a basis for subsequent clinical trials of ADV-ApoA1 in the treatment of TNBC.

Huayu Qutan Recipe promotes lipophagy and cholesterol efflux through the mTORC1/TFEB/ABCA1-SCARB1 signal axis.

This study aims to investigate the mechanism of the anti-atherosclerosis effect of Huayu Qutan Recipe (HYQT) on the inhibition of foam cell formation. Invivo, the mice were randomly divided into three groups: CTRL group, MOD group and HYQT group. The HYQT group received HYQT oral administration twice a day (20.54 g/kg/d), and the plaque formation in ApoE-/- mice was observed using haematoxylin-eosin (HE) staining and oil red O (ORO) staining. The co-localization of aortic macrophages and lipid droplets (LDs) was examined using fluorescent labelling of CD11b and BODIPY fluorescence probe. Invitro, RAW 264.7 cells were exposed to 50 μg/mL ox-LDL for 48 h and then treated with HYQT for 24 h. The accumulation of LDs was evaluated using ORO and BODIPY. Cell viability was assessed using the CCK-8 assay. The co-localization of LC3b and BODIPY was detected via immunofluorescence and fluorescence probe. LysoTracker Red and BODIPY 493/503 were used as markers for lysosomes and LDs, respectively. Autophagosome formation were observed via transmission electron microscopy. The levels of LC3A/B II/LC3A/B I, p-mTOR/mTOR, p-4EBP1/4EBP1, p-P70S6K/P70S6K and TFEB protein level were examined via western blotting, while SQSTM1/p62, Beclin1, ABCA1, ABCG1 and SCARB1 were examined via qRT-PCR and western blotting. The nuclear translocation of TFEB was detected using immunofluorescence. The components of HYQT medicated serum were determined using Q-Orbitrap high-resolution MS analysis. Molecular docking was employed to identify the components of HYQT medicated serum responsible for the mTOR signalling pathway. The mechanism of taurine was illustrated. HYQT has a remarkable effect on atherosclerotic plaque formation and blood lipid level in ApoE-/- mice. HYQT decreased the co-localization of CD11b and BODIPY. HYQT (10% medicated serum) reduced the LDs accumulation in RAW 264.7 cells. HYQT and RAPA (rapamycin, a mTOR inhibitor) could promote cholesterol efflux, while chloroquine (CQ, an autophagy inhibitor) weakened the effect of HYQT. Moreover, MHY1485 (a mTOR agonist) also mitigated the effects of HYQT by reduced cholesterol efflux. qRT-PCR and WB results suggested that HYQT improved the expression of the proteins ABCA1, ABCG1 and SCARB1.HYQT regulates ABCA1 and SCARB1 protein depending on the mTORC1/TFEB signalling pathway. However, the activation of ABCG1 does not depend on this pathway. Q-Orbitrap high-resolution MS analysis results demonstrated that seven core compounds have good binding ability to the mTOR protein. Taurine may play an important role in the mechanism regulation. HYQT may reduce cardiovascular risk by promoting cholesterol efflux and degrading macrophage-derived foam cell formation. It has been observed that HYQT and ox-LDL regulate lipophagy through the mTOR/TFEB signalling pathway, rather than the mTOR/4EBP1/P70S6K pathway. Additionally, HYQT is found to regulate cholesterol efflux through the mTORC1/TFEB/ABCA1-SCARB1 signal axis, while taurine plays a significant role in lipophagy.

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.