Cyclophilin A accelerates SiO2-induced macrophage foaming.

Silicosis is a common occupational disease characterized by lung inflammation, fibrosis and pulmonary dysfunction caused by long-term inhalation of free SiO2. Cell foaming and the change of CyPA have been observed in SiO2-induced macrophages, but the specific mechanism of CyPA in SiO2-induced foam cells remains poorly understood. The purpose of this study is to explore the mechanism of CyPA in SiO2-induced macrophage foaming and its effect on silicosis. We found that overexpression of CyPA promoted the macrophage foaming and the expression of COL I and α-SMA, while silencing CyPA inhibites the macrophage foaming and the expression of COL I and α-SMA. After blocking the expression of CD36 on the basis of overexpression CyPA, we found it inhibites the macrophage foaming. In conclusion, CyPA can affect the foaming of macrophages and may participate in silicosis fibrosis.

Low LAL (Lysosomal Acid Lipase) Expression by Smooth Muscle Cells Relative to Macrophages as a Mechanism for Arterial Foam Cell Formation.

[Figure: see text].

A new activator of esterase D decreases blood cholesterol level through ESD/JAB1/ABCA1 pathway.

Excessively high cholesterol content in the blood leads to nonalcohol fatty liver disease (NAFLD) and arteriosclerosis. Although there are increasing publications and patent applications to lower blood cholesterol with small chemical molecules, limited effective drugs can be available in clinic. It is necessary to uncover new targets and drugs to alleviate high cholesterol. Esterase D (ESD) is abundant in liver and it remains unknown about its role in cholesterol metabolism. Here we reported that small chemical molecule fluorescigenic pyrazoline derivative 5 (FPD5), a new ESD activator, could effectively reverse high blood cholesterol level and prevent fatty liver and arteriosclerosis in apoE-/- mice fed the high-fat diet. We also observed that FPD5 could reduce oxidized low density lipoprotein (oxLDL)-induced formation of foam cells. To further investigate the mechanism of FPD5 action on blood cholesterol modulation, we found that ESD trigged by FPD5 was aggregated in lysosome and interacted with Jun activation domain binding protein 1 (JAB1). ESD served as a deacetylase to remove Thr89 acetylation of JAB1 and increased its activity; thus, promoting the ATP-binding cassette transporters A1 (ABCA1) to accelerate cholesterol efflux. Our findings demonstrate that FPD5 decreases blood cholesterol level to ameliorate NAFLD and arteriosclerosis through ESD/JAB1/ABCA1 pathway, and ESD functions as a novel nonclassical deacetylase that hydrolyzes serine/threonine acetyl group. Our findings not only highlight that FPD5 may be a pioneer drug for alleviating blood cholesterol but also indicate that ESD is a potential drug target that promotes cholesterol metabolism.

A-family anti-inflammatory cyclopentenone prostaglandins: A novel class of non-statin inhibitors of HMG-CoA reductase.

Disruption of the intracellular lipid balance leading to cholesterol accumulation is one of the features of cells that participate in the development of atherosclerotic lesions. Evidence form our laboratory indicates that anti-inflammatory cyclopentenone prostaglandins (cyPGs) of A- and J-family deviate lipid metabolism from the synthesis of cholesterol and cholesteryl esters to the synthesis of phospholipids in foam-cell macrophages. cyPGs possessing an α,ß-unsaturated cyclopentane ring are highly electrophilic substances able to promptly react with reactive cysteines of intracellular molecules through Michael addition. On the other hand, HMG-CoA reductase (HMGCR), the enzyme responsible for the rate-limiting step in cholesterol biosynthesis, presents critically reactive cysteines at the entry of catalytic domain, particularly Cys561, that could be target of cyPG inhibition. In the present study, we showed that cyPGs (but not other non-α,ß-unsaturated PGs) physically interact with HMGCR, in a dithiothreitol- and ß-mercaptoethanol-sensitive way, and block the activity of the catalytic subunit of the enzyme (IC50 for PGA2 = 0.17 µM). PGA2 inhibits HMGCR activity in cultured rat and human macrophages/macrophage-foam cells and leads to enhanced expression of HMGCR protein, as observed with statins. In cell culture models, PGA2 effectively inhibits the reductase at non-toxic doses (e.g., 1 µM) that block cell proliferation thus suggesting that part of the well-known antiproliferative effect of PGA2 may be due to its ability of blocking HMGCR activity, as cells cannot proliferate without a robust cholesterogenesis. Therefore, besides the powerfully anti-inflammatory and antiproliferative effects, the anticholesterogenic effects of PGA2 should be exploited in atherosclerosis therapeutics.

Atorvastatin Enhances Foam Cell Lipophagy and Promotes Cholesterol Efflux Through the AMP-Activated Protein Kinase/Mammalian Target of Rapamycin Pathway.

ABSTRACT: Foam cells are the main pathological components of atherosclerosis. Therapies reducing foam cell formation can effectively prevent atherosclerotic diseases and cardiovascular events. Beyond lowering plasma cholesterol levels, the pleiotropic functions of statins in atherosclerosis have not been fully elucidated. In the present study, atorvastatin reduced cholesterol content and increased cholesterol efflux from foam cells in a concentration-dependent manner. Atorvastatin (10 µM) inhibited foam cell formation within 48 hours. Furthermore, we found that atorvastatin inhibited foam cell formation by promoting lipophagy, which was manifested by increased autophagy-related gene 5 (Atg5) expression, elevated ratio of microtubule-associated protein1 light chain 3 (LC3) II to LC3I, reduced p62 expression, and increased LC3 and lipid droplets colocalization in foam cells treated with atorvastatin. The autophagy inducer, rapamycin (Rap), did not increase the lipophagy enhancement effect of atorvastatin, but the autophagy inhibitor, 3-methyladenine, suppressed the effect of atorvastatin on Atg5 expression and the LC3II/LC3I ratio, as well as the increased p62 expression, suppressed lipophagy, attenuated cholesterol efflux and increased cholesterol content in foam cells. Further analysis revealed that atorvastatin promoted lipophagy by upregulating adenosine 5'-monophosphate-activated protein kinase (AMPK) phosphorylation, and downregulating mammalian target of rapamycin phosphorylation, whereas the AMPK inhibiter, compound C, attenuated these effects. In conclusion, atorvastatin reduced lipid accumulation and promoted cholesterol efflux by enhancing lipophagy in foam cells and thereby inhibited foam cell formation. The enhanced lipophagy of foam cells was exerted through the AMPK/mammalian target of rapamycin signaling pathway.

PCSK9 and LRP5 in macrophage lipid internalization and inflammation.

AIMS: Atherosclerosis, the leading cause of cardiovascular diseases, is driven by high blood cholesterol levels and chronic inflammation. Low-density lipoprotein receptors (LDLR) play a critical role in regulating blood cholesterol levels by binding to and clearing LDLs from the circulation. The disruption of the interaction between proprotein convertase subtilisin/kexin 9 (PCSK9) and LDLR reduces blood cholesterol levels. It is not well known whether other members of the LDLR superfamily may be targets of PCSK9. The aim of this work was to determine if LDLR-related protein 5 (LRP5) is a PCSK9 target and to study the role of PCSK9 and LRP5 in foam cell formation and lipid accumulation. METHODS AND RESULTS: Primary cultures of human inflammatory cells (monocytes and macrophages) were silenced for LRP5 or PCSK9 and challenged with LDLs. We first show that LRP5 is needed for macrophage lipid uptake since LRP5-silenced macrophages show less intracellular CE accumulation. In macrophages, internalization of LRP5-bound LDL is already highly evident after 5 h of LDL incubation and lasts up to 24 h; however, in the absence of both LRP5 and PCSK9, there is a strong reduction of CE accumulation indicating a role for both proteins in lipid uptake. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in lipid-loaded macrophages. Finally, PCSK9 participates in TLR4/NFkB signalling; a decreased TLR4 protein expression levels and a decreased nuclear translocation of NFκB were detected in PCSK9 silenced cells after lipid loading, indicating a downregulation of the TLR4/NFκB pathway. CONCLUSION: Our results show that both LRP5 and PCSK9 participate in lipid uptake in macrophages. In the absence of LRP5, there is a reduced release of PCSK9 indicating that LRP5 also participates in the mechanism of release of soluble PCSK9. Furthermore, PCSK9 up-regulates TLR4/NFκB favouring inflammation.

Foam Cell Induction Activates AMPK But Uncouples Its Regulation of Autophagy and Lysosomal Homeostasis.

The dysregulation of macrophage lipid metabolism drives atherosclerosis. AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics and plays essential roles regulating macrophage lipid dynamics. Here, we investigated the consequences of atherogenic lipoprotein-induced foam cell formation on downstream immunometabolic signaling in primary mouse macrophages. A variety of atherogenic low-density lipoproteins (acetylated, oxidized, and aggregated forms) activated AMPK signaling in a manner that was in part due to CD36 and calcium-related signaling. In quiescent macrophages, basal AMPK signaling was crucial for maintaining markers of lysosomal homeostasis as well as levels of key components in the lysosomal expression and regulation network. Moreover, AMPK activation resulted in targeted upregulation of members of this network via transcription factor EB. However, in lipid-induced macrophage foam cells, neither basal AMPK signaling nor its activation affected lysosomal-associated programs. These results suggest that while the sum of AMPK signaling in cultured macrophages may be anti-atherogenic, atherosclerotic input dampens the regulatory capacity of AMPK signaling.

Role of Macrophages and RhoA Pathway in Atherosclerosis.

The development, progression, or stabilization of the atherosclerotic plaque depends on the pro-inflammatory and anti-inflammatory macrophages. The influx of the macrophages and the regulation of macrophage phenotype, inflammatory or anti-inflammatory, are controlled by the small GTPase RhoA and its downstream effectors. Therefore, macrophages and the components of the RhoA pathway are attractive targets for anti-atherosclerotic therapies, which would inhibit macrophage influx and inflammatory phenotype, maintain an anti-inflammatory environment, and promote tissue remodeling and repair. Here, we discuss the recent findings on the role of macrophages and RhoA pathway in the atherosclerotic plaque formation and resolution and the novel therapeutic approaches.

USP10 deletion inhibits macrophage-derived foam cell formation and cellular-oxidized low density lipoprotein uptake by promoting the degradation of CD36.

Foam cell formation process is involved in the pathogenesis of atherosclerosis (AS). Activation of this biological process depends on lipid uptake by scavenger receptors, such as CD36, SR-A and SR-B1. Among these receptors, CD36 is the principal one because it dominates roughly 50% lipid uptake in monocytes. In this study, our western blotting and RT-qPCR assays revealed that USP10 inhibition promotes the degradation of CD36 protein but does not change its mRNA level. In addition, Co-IP results showed that USP10 interacts with CD36 and stabilizes CD36 protein by cleaving poly-ubiquitin on CD36. Significantly, USP10 promotes foam cell formation. Immunofluorescence and Oil red O staining assays show that inhibition or knockdown of USP10 suppresses lipid uptake and foam cell formation by macrophages. In conclusion, USP10 promotes the development and progression of atherosclerosis through stabilizing CD36 protein expression. The regulation of USP10-CD36 may provide a significant therapeutic scheme in atherosclerosis.

Loss of MLKL (Mixed Lineage Kinase Domain-Like Protein) Decreases Necrotic Core but Increases Macrophage Lipid Accumulation in Atherosclerosis.

OBJECTIVES: During the advancement of atherosclerosis, plaque cellularity is governed by the influx of monocyte-derived macrophages and their turnover via apoptotic and nonapoptotic forms of cell death. Previous reports have demonstrated that programmed necrosis, or necroptosis, of plaque macrophages contribute to necrotic core formation. Knockdown or inhibition of the necrosome components RIPK1 (receptor-interacting protein kinase 1) and RIPK3 (receptor-interacting protein kinase 3) slow atherogenesis, and activation of the terminal step of necroptosis, MLKL (mixed lineage kinase domain-like protein), has been demonstrated in advanced human atherosclerotic plaques. However, whether MLKL directly contributes to lesion development and necrotic core formation has not been investigated. Approaches and Results: MLKL expression was knocked down in atherogenic Apoe-knockout mice via the administration of antisense oligonucleotides. During atherogenesis, Mlkl knockdown decreased both programmed cell death and the necrotic core in the plaque. However, total lesion area remained unchanged. Furthermore, treatment with the MLKL antisense oligonucleotide unexpectedly reduced circulating cholesterol levels compared with control antisense oligonucleotide but increased the accumulation of lipids within the plaque and in vitro in macrophage foam cells. MLKL colocalized with the late endosome and multivesicular bodies in peritoneal macrophages incubated with atherogenic lipoproteins. Transfection with MLKL antisense oligonucleotide increased lipid localization with the multivesicular bodies, suggesting that upon Mlkl knockdown, lipid trafficking becomes defective leading to enhanced lipid accumulation in macrophages. CONCLUSIONS: These studies confirm the requirement for MLKL as the executioner of necroptosis, and as such a significant contributor to the necrotic core during atherogenesis. We also identified a previously unknown role for MLKL in regulating endosomal trafficking to facilitate lipid handling in macrophages during atherogenesis.

Artemisinin Attenuated Atherosclerosis in High-Fat Diet-Fed ApoE-/- Mice by Promoting Macrophage Autophagy Through the AMPK/mTOR/ULK1 Pathway.

Artemisinin is an endoperoxide sesquiterpene lactone from Artemisia annua L with multiple beneficial effects, including anti-inflammation, antioxidant, and vascular protection. Recent studies have found that inflammation along with autophagy deficiency in macrophages is the possible reason for foam cell accumulation in the intima, which leads to atherosclerotic plaque formation. The primary aims of this study were to explore the inhibiting effect of artemisinin on atherosclerosis in high-fat diet-fed ApoE mice and investigate the probable mechanism. Artemisinin (50 and 100 mg/kg, intragastric administration) treatment effectively inhibited foamy macrophage transformation and decreased atherosclerotic plaque formation in atherosclerotic mice. Moreover, artemisinin promoted AMP-activated protein kinase (AMPK) activation, inhibited mammalian target of rapamycin (mTOR) and uncoordinated-51-like kinase 1 (ULK1) phosphorylation, and increased LC-3II accumulation and P62 degradation, thereby enhancing macrophage autophagy. Besides, the inhibiting effect of artemisinin on mTOR and ULK1 phosphorylation could be abrogated by AMPK knockdown, suggesting AMPK was the essential target of artemisinin on promoting macrophage autophagy. Our study indicated that artemisinin alleviated atherosclerotic lesions by accelerating macrophage autophagy through the AMPK/mTOR/ULK1 pathway.

Berberine Attenuates Cholesterol Accumulation in Macrophage Foam Cells by Suppressing AP-1 Activity and Activation of the Nrf2/HO-1 Pathway.

Atherosclerosis is a chronic inflammation condition resulting from the interaction between lipoproteins, monocyte-derived macrophages, T lymphocytes, and other cellular elements in the arterial wall. Macrophage-derived foam cells play a key role in both early and advanced stage of atherosclerosis. Previous studies have shown that berberine could inhibit foam cell formation and prevent experimental atherosclerosis. However, its underlying molecular mechanisms have not been fully clarified. In this study, we explored the cholesterol-lowering effects of berberine in macrophage-derived foam cells and investigated its possible mechanisms in prevention and treatment of atherosclerosis. Here, we demonstrated that berberine could inhibit atherosclerosis in apolipoprotein E-deficient mice and induce cholesterol reduction as well as decrease the content of macrophages. Berberine can regulate oxLDL uptake and cholesterol efflux, thus suppresses foam cell formation. Mechanisms study showed that berberine can suppress scavenger receptor expression via inhibiting the activity of AP-1 and upregulate ATP-binding cassette transporter via activating Nrf2/HO-1 signaling in human macrophage. In summary, berberine significantly inhibits atherosclerotic disease development by regulating lipid homeostasis and suppressing macrophage foam cell formation.

miR-33-5p knockdown attenuates abdominal aortic aneurysm progression via promoting target adenosine triphosphate-binding cassette transporter A1 expression and activating the PI3K/Akt signaling pathway.

PURPOSE: The aim of this study was to investigate the role of miR-33-5p in abdominal aortic aneurysm progression, which regulated adenosine triphosphate-binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux and lipid accumulation in THP-1 macrophage-derived foam cells through the PI3K/Akt pathway. METHODS: Quantitative reverse transcription polymerase chain reaction was used to evaluate the expression level of miR-33-5p and ABCA1 mRNA in abdominal aortic aneurysm patient and normal person tissues. The relationship between miR-33-5p and ABCA1 was examined by dual luciferase report assay. High-performance liquid chromatography was used to evaluate the levels of cholesterol contents. Cholesterol efflux detection was performed by liquid scintillator. The expression of inflammatory cytokines was detected by quantitative reverse transcription polymerase chain reaction. Western blot was applied to determine the expression levels of ABCA1, PI3K (p-PI3K), and Akt (p-Akt). RESULTS: The quantitative reverse transcription polymerase chain reaction analysis results revealed miR-33-5p overexpression in abdominal aortic aneurysm tissues, but the expression level of ABCA1 was lower in abdominal aortic aneurysm tissues than non-abdominal aortic aneurysm tissues. Subsequently, the dual luciferase report gene assay confirmed that ABCA1 was a target of miR-33-5p, and miR-33-5p-negative regulated ABCA1 expression. Moreover, the expression levels of p-PI3K, p-Akt, and ABCA1 were decreased in THP-1 cell transferred with ABCA1 siRNA, but knockdown of miR-33-5p had an opposite effect. Furthermore, knockdown of miR-33-5p decreased the expression of MMP-2, MMP-9, TNF-α, total cellular cholesterol, and promoted cholesterol efflux in THP-1-derived foam cells. Importantly, LY294002 (PI3K inhibitor) or si-ABCA1 completely inhibited the stimulatory effects of miR-33-5p inhibitor. CONCLUSION: This study has found that knockdown of miR-33-5p induced ABCA1 expression and promoted inflammatory cytokines and cholesterol efflux likely via activating the PI3K/Akt signaling pathway.

Inhibition of Soluble Epoxide Hydrolase in Macrophages Ameliorates the Formation of Foam Cells　- Role of Heme Oxygenase-1.

BACKGROUND: Accumulation of foam cells in the neointima represents an early stage of atherosclerosis. 1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl) urea (TPPU), a novel soluble epoxide hydrolase inhibitor (sEHi), effectively elevates epoxyeicosatrienoic acid (EET) levels. The effects of EETs on macrophages foam cells formation are poorly understood.Methods and Results:Incubation of foam cells with TPPU markedly ameliorate cholesterol deposition in oxidized low-density lipoprotein (oxLDL)-loaded macrophages by increasing the levels of EETs. Notably, TPPU treatment significantly inhibits oxLDL internalization and promotes cholesterol efflux. The elevation of EETs results in a decrease of class A scavenger receptor (SR-A) expression via downregulation of activator protein 1 (AP-1) expression. Additionally, TPPU selectively increases protein but not the mRNA level of ATP-binding cassette transporter A1 (ABCA1) through the reduction of calpain activity that stabilizes the protein. Moreover, TPPU treatment reduces the cholesterol content of macrophages and inhibits atherosclerotic plaque formation in apolipoprotein E-deficient mice. These changes induced by TPPU are dependent on heme oxygenase-1 (HO-1) activation. CONCLUSIONS: The present study findings elucidate a precise mechanism of regulating cholesterol uptake and efflux in macrophages, which involves the prevention of atherogenesis by increasing the levels of EETs with TPPU.

Lysophosphatidic acid decreased macrophage foam cell migration correlated with downregulation of fucosyltransferase 8 via HNF1α.

BACKGROUND AND AIMS: Aberrant fucosylation, such as α-1,6 fucosylation catalyzed by fucosyltransferase 8 (Fut8), is associated with reduced cell migration and is responsible for cholesterol-enriched foam cell accumulation in the intima in the early stage of atherosclerosis. The current study evaluated the impact of glycosyltransferases on foam cell migration induced by lysophosphatidic acid (LPA) and its potential mechanism. METHODS: The mobility of foam cells was evaluated via transwell and scratch assays. The expression of Fut8 and α-1,6 fucosylation of proteins were assessed by RT-PCR, Western blotting, etc. Overexpression of Fut8 was used to explore the direct relationship between Fut8 and foam cell migration. Dual luciferase reporter assay was performed to determine whether the regulation of Fut8 by LPA occurred at the transcriptional level. Binding of hepatocyte nuclear factor 1-alpha (HNF1α) to the Fut8 promoter was assessed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. RESULTS: We found that the migration capacity of foam cells induced by LPA was significantly decreased. Fut8 and α-1,6 fucosylation showed the most obvious decline after treatment with 200 µM LPA for 24 h. Overexpression of Fut8 was able to restore the foam cell migration capacity. Another important finding was that the LPA1 and LPA3 (LPA1,3) receptors were involved in the regulation of Fut8. It is interesting to note that LPA led to a decrease in Fut8 gene transcription activity, and HNF1α transcription factor played a positive role in downregulation of Fut8 promoter activity. CONCLUSIONS: Our results strongly indicated that the LPA-LPA1, 3 receptor-HNF1α pathway is involved in the downregulation of Fut8, leading to diminished foam cell migration.

The phosphatase activity of soluble epoxide hydrolase regulates ATP-binding cassette transporter-A1-dependent cholesterol efflux.

The contribution of soluble epoxide hydrolase (sEH) to atherosclerosis has been well defined. However, less is understood about the role of sEH and its underlying mechanism in the cholesterol metabolism of macrophages. The expression of sEH protein was increased in atherosclerotic aortas of apolipoprotein E-deficient mice, primarily in macrophage foam cells. Oxidized low-density lipoprotein (oxLDL) increased sEH expression in macrophages. Genetic deletion of sEH (sEH-/- ) in macrophages markedly exacerbated oxLDL-induced lipid accumulation and decreased the expression of ATP-binding cassette transporters-A1 (ABCA1) and apolipoprotein AI-dependent cholesterol efflux following oxLDL treatment. The down-regulation of ABCA1 in sEH-/- macrophages was due to an increase in the turnover rate of ABCA1 protein but not in mRNA transcription. Inhibition of phosphatase activity, but not hydrolase activity, of sEH decreased ABCA1 expression and cholesterol efflux following oxLDL challenge, which resulted in increased cholesterol accumulation. Additionally, oxLDL increased the phosphatase activity, promoted the sEH-ABCA1 complex formation and decreased the phosphorylated level of ABCA1 at threonine residues. Overexpression of phosphatase domain of sEH abrogated the oxLDL-induced ABCA1 phosphorylation and further increased ABCA1 expression and cholesterol efflux, leading to the attenuation of oxLDL-induced cholesterol accumulation. Our findings suggest that the phosphatase domain of sEH plays a crucial role in the cholesterol metabolism of macrophages.

Ablation of Myeloid ADK (Adenosine Kinase) Epigenetically Suppresses Atherosclerosis in ApoE-/- (Apolipoprotein E Deficient) Mice.

Objective- Monocyte-derived foam cells are one of the key players in the formation of atherosclerotic plaques. Adenosine receptors and extracellular adenosine have been demonstrated to modulate foam cell formation. ADK (adenosine kinase) is a major enzyme regulating intracellular adenosine levels, but its functional role in myeloid cells remains poorly understood. To enhance intracellular adenosine levels in myeloid cells, ADK was selectively deleted in novel transgenic mice using Cre-LoxP technology, and foam cell formation and the development of atherosclerotic lesions were determined. Approach and Results- ADK was upregulated in macrophages on ox-LDL (oxidized low-density lipoprotein) treatment in vitro and was highly expressed in foam cells in atherosclerotic plaques. Atherosclerotic mice deficient in ADK in myeloid cells were generated by breeding floxed ADK (ADKF/F) mice with LysM-Cre (myeloid-specific Cre recombinase expressing) mice and ApoE-/- (apolipoprotein E deficient) mice. Mice absent ADK in myeloid cells exhibited much smaller atherosclerotic plaques compared with controls. In vitro assays showed that ADK deletion or inhibition resulted in increased intracellular adenosine and reduced DNA methylation of the ABCG1 (ATP-binding cassette transporter G1) gene. Loss of methylation was associated with ABCG1 upregulation, enhanced cholesterol efflux, and eventually decreased foam cell formation. Conclusions- Augmentation of intracellular adenosine levels through ADK knockout in myeloid cells protects ApoE-/- mice against atherosclerosis by reducing foam cell formation via the epigenetic regulation of cholesterol trafficking. ADK inhibition is a promising approach for the treatment of atherosclerotic diseases.

Myeloid Kdm6b deficiency results in advanced atherosclerosis.

BACKGROUND AND AIMS: Atherosclerosis is a lipid-driven chronic inflammatory disorder of the arteries, and monocytes and macrophages play a central role in this process. Within the atherosclerotic lesion, macrophages can scavenge modified lipids and become the so-called foam cells. We previously reported that the epigenetic enzyme Kdm6b (also known as Jmjd3) controls the pro-fibrotic transcriptional profile of peritoneal foam cells. Given the importance of these cells in atherosclerosis, we now studied the effect of myeloid Kdm6b on disease progression. METHODS: Bone marrow of myeloid Kdm6b deficient (Kdm6bdel) mice or wild type littermates (Kdm6bwt) was transplanted to lethally irradiated Ldlr-/- mice fed a high fat diet for 9 weeks to induce atherosclerosis. RESULTS: Lesion size was similar in Kdm6bwt and Kdm6bdel transplanted mice. However, lesions of Kdm6bdel mice contained more collagen and were more necrotic. Pathway analysis on peritoneal foam cells showed that the pathway involved in leukocyte chemotaxis was most significantly upregulated. Although macrophage and neutrophil content was similar after 9 weeks of high fat diet feeding, the relative increase in collagen content and necrosis revealed that atherosclerotic lesions in Kdm6bdel mice progress faster. CONCLUSION: Myeloid Kdm6b deficiency results in more advanced atherosclerosis.

Lyso-DGTS lipid isolated from microalgae enhances PON1 activities in vitro and in vivo, increases PON1 penetration into macrophages and decreases cellular lipid accumulation.

High-density lipoprotein (HDL) plays an important role in preventing atherosclerosis. The antioxidant effect of HDL is mostly associated with paraoxonase 1 (PON1) activity. Increasing PON1 activity using nutrients might improve HDL function and quality and thus, decrease atherosclerotic risk. We previously isolated and identified a novel active compound, lyso-DGTS (C20:5,0) from Nannochloropsis sp. ethanol extract. In the present study, its effect on PON1 activities was examined and the mechanism by which the compound affects PON1 activity was explored. Lyso-DGTS elevated recombinant PON1 (rePON1) lactonase and esterase activities in a dose- and time-responsive manner, and further stabilized and preserved rePON1 lactonase activity. Incubation of lyso-DGTS with human serum for 4 h at 37 °C also increased PON1 lactonase activity in a dose-responsive manner. Using tryptophan-fluorescence-quenching assay, lyso-DGTS was found to interact with rePON1 spontaneously with negative free energy (ΔG = -22.87 kJ mol-1 at 25 °C). Thermodynamic parameters and molecular modeling calculations showed that the main interaction of lyso-DGTS with the enzyme is through a hydrogen bond with supporting van der Waals interactions. Furthermore, lyso-DGTS significantly increased rePON1 influx into macrophages and prevented lipid accumulation in macrophages stimulated with oxidized low-density lipid dose-dependently. In vivo supplementation of lyso-DGTS to the circulation of mice fed a high-fat diet via osmotic mini-pumps implanted subcutaneously significantly increased serum PON1 lactonase activity and decreased serum glucose concentrations to the level of mice fed a normal diet. Our findings suggest a beneficial effect of lyso-DGTS on increasing PON1 activity and thus, improving HDL quality and atherosclerotic risk factors. © 2018 BioFactors, 44(3):299-310, 2018.

Roles for endothelial cell and macrophage Gch1 and tetrahydrobiopterin in atherosclerosis progression.

Aims: GTP cyclohydrolase I catalyses the first and rate-limiting reaction in the synthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases (NOS). Both eNOS and iNOS have been implicated in the progression of atherosclerosis, with opposing effects in eNOS and iNOS knockout mice. However, the pathophysiologic requirement for BH4 in regulating both eNOS and iNOS function, and the effects of loss of BH4 on the progression of atherosclerosis remains unknown. Methods and results: Hyperlipidemic mice deficient in Gch1 in endothelial cells and leucocytes were generated by crossing Gch1fl/flTie2cre mice with ApoE-/- mice. Deficiency of Gch1 and BH4 in endothelial cells and myeloid cells was associated with mildly increased blood pressure. High fat feeding for 6 weeks in Gch1fl/flTie2CreApoE-/- mice resulted in significantly decreased circulating BH4 levels, increased atherosclerosis burden and increased plaque macrophage content. Gch1fl/flTie2CreApoE-/- mice showed hallmarks of endothelial cell dysfunction, with increased aortic VCAM-1 expression and decreased endothelial cell dependent vasodilation. Furthermore, loss of BH4 from pro-inflammatory macrophages resulted in increased foam cell formation and altered cellular redox signalling, with decreased expression of antioxidant genes and increased reactive oxygen species. Bone marrow chimeras revealed that loss of Gch1 in both endothelial cells and leucocytes is required to accelerate atherosclerosis. Conclusion: Both endothelial cell and macrophage BH4 play important roles in the regulation of NOS function and cellular redox signalling in atherosclerosis.