BACKGROUND: Exosome-miR-146a is significantly increased in patients with Atherosclerosis (AS), but its mechanism and effect on AS have not been fully elucidated.
OBJECTIVE: To explore the change rule and mechanism of exosomes release, and the role and molecular mechanism of exosome-miR-146a in AS.
METHODS: We isolated and identified exosomes from THP-1 macrophages after treating them with ox-LDL. Then used co-immunoprecipitation and silver staining to identify the proteins involved in regulating exosome release. PKH67 was used to label exosomes to confirm that cells can absorb them, and then co-culture with HVSMCs for cell proliferation and migration detection. The target genes of miR-146a were screened and identified through bioinformatics and luciferase activity assay, and the expression of miR-146a and related proteins was detected through qRT-PCR and Western blot in HUVECs. An AS model in LDLR-/- mice induced by a high-fat diet was developed to investigate the impact of exosome-miR-146a on AS.
RESULTS: The results showed that experimental foam cells from AS showed higher expression of miR-146a. It was observed that NMMHC IIA and HSP70 interacted to regulate the release of exosomes. And HUVECs can absorb exosomes derived from macrophages. In addition, we also found that miR-146a directly targeted the SMAD4 gene to modulate the p38 MAPK signaling pathway, thereby mediating HUVECs damage. Furthermore, exosome-miR-146a induced abnormal proliferation and migration of HVSMCs. The expression of miR-146a was significantly reduced in miR-146a-mimics mice and increased in miR-146a inhibitor mice whereas the inhibition of miR-146a effectively reduced while increasing miR-146a worsened AS in mice.
CONCLUSIONS: Our findings expressed the potential of miR-146a as a favorable therapeutic target for AS, however, further exploration is suggestive for deep understanding of the mechanisms regulating exosome-miR-146a release in vivo and to develop effective therapeutic strategies involving miR-146a.

Atherosclerotic plaque formation is largely attributed to the impaired efferocytosis, which is known to be associated with the pathologic upregulation of cluster of differentiation 47 (CD47), a key antiphagocytic molecule. By gene expression omnibus (GEO) datasets analysis, we identified that four miRNAs are aberrantly downregulated in atherosclerosis, coronary artery disease, and obesity. Of them, hsa-miR-299-3p (miR-299-3p) was predicted to target the 3\'UTR of human CD47 mRNA by bioinformatics analysis. Further, we demonstrated that miR-299-3p negatively regulates CD47 expression by binding to the target sequence \"CCCACAU\" in the 3\'UTR of CD47 mRNA through luciferase reporter assay and site-directed mutagenesis. Additionally, we found that miR-299-3p was downregulated by ~32% in foam cells in response to oxidized low-density lipoprotein (ox-LDL) stimulation, thus upregulating CD47 and contributing to the impaired efferocytosis. Whereas, restoration of miR-299-3p reversed the ox-LDL-induced upregulation of CD47, thereby facilitating efferocytosis. In high-fat diet (HFD) fed ApoE-/- mice, we discovered that miR-299-3p was downregulated thus leading to upregulation of CD47 in abdominal aorta. Conversely, miR-299-3p restoration potently suppressed HFD-induced upregulation of CD47 and promoted phagocytosis of foam cells by macrophages in atherosclerotic plaques, thereby reducing necrotic core, increasing plaque stability, and mitigating atherosclerosis. Conclusively, we identify miR-299-3p as a negative regulator of CD47, and reveal a molecular mechanism whereby the ox-LDL-induced downregulation of miR-299-3p leads to the upregulation of CD47 in foam cells thus contributing to the impaired efferocytosis in atherosclerosis, and propose miR-299-3p can potentially serve as an inhibitor of CD47 to promote efferocytosis and ameliorate atherosclerosis.

The rising incidences of atherosclerosis have necessitated efforts to identify novel targets for therapeutic interventions. In the present study, we observed increased expression of the mechanosensitive calcium channel Piezo1 transcript in mouse and human atherosclerotic plaques, correlating with infiltration of PIEZO1-expressing macrophages. In vitro administration of Yoda1, a specific agonist for PIEZO1, led to increased foam cell apoptosis and enhanced phagocytosis by macrophages. Mechanistically, PIEZO1 activation resulted in intracellular F-actin rearrangement, elevated mitochondrial ROS levels and induction of mitochondrial fragmentation upon PIEZO1 activation, as well as increased expression of anti-inflammatory genes. In vivo, ApoE-/- mice treated with Yoda1 exhibited regression of atherosclerosis, enhanced stability of advanced lesions, reduced plaque size and necrotic core, increased collagen content, and reduced expression levels of inflammatory markers. Our findings propose PIEZO1 as a novel and potential therapeutic target in atherosclerosis.

The formation of macrophage-derived foam cells has been recognized as the pathological hallmark of atherosclerotic diseases. However, the pathological evolution dynamics and underlying regulatory mechanisms remain largely unknown. Herein, we introduce a single-particle rotational microrheology method for pathological staging of macrophage foaming and antiatherosclerotic explorations by probing the dynamic changes of lysosomal viscous feature over the pathological evolution progression. The principle of this method involves continuous monitoring of out-of-plane rotation-caused scattering brightness fluctuations of the gold nanorod (AuNR) probe-based microrheometer and subsequent determination of rotational relaxation time to analyze the viscous feature in macrophage lysosomes. With this method, we demonstrated the lysosomal viscous feature as a robust pathological reporter and uncovered three distinct pathological stages underlying the evolution dynamics, which are highly correlated with a pathological stage-dependent activation of the NLRP3 inflammasome-involved positive feedback loop. We also validated the potential of this positive feedback loop as a promising therapeutic target and revealed the time window-dependent efficacy of NLRP3 inflammasome-targeted drugs against atherosclerotic diseases. To our knowledge, the pathological staging of macrophage foaming and the pathological stage-dependent activation of the NLRP3 inflammasome-involved positive feedback mechanism have not yet been reported. These findings provide insights into in-depth understanding of evolutionary features and regulatory mechanisms of macrophage foaming, which can benefit the analysis of effective therapeutical drugs as well as the time window of drug treatment against atherosclerotic diseases in preclinical studies.

Foam cells in atheroma are engorged with lipid droplets (LDs) that contain esters of regulatory lipids whose metabolism remains poorly understood. LD-associated hydrolase (LDAH) has a lipase structure and high affinity for LDs of foam cells. Using knockout and transgenic mice of both sexes, here we show that LDAH inhibits atherosclerosis development and promotes stable lesion architectures. Broad and targeted lipidomic analyzes of primary macrophages and comparative lipid profiling of atheroma identified a broad impact of LDAH on esterified sterols, including natural liver X receptor (LXR) sterol ligands. Transcriptomic analyzes coupled with rescue experiments show that LDAH modulates the expression of prototypical LXR targets and leads macrophages to a less inflammatory phenotype with a profibrotic gene signature. These studies underscore the role of LDs as reservoirs and metabolic hubs of bioactive lipids, and suggest that LDAH favorably modulates macrophage activation and protects against atherosclerosis via lipolytic mobilization of regulatory sterols.

Cardiovascular diseases (CVDs) linked to atherosclerosis remains the leading cause of death worldwide. Atherosclerosis is primarily caused by the accumulation of oxidized forms of low density lipoprotein (LDL) in macrophages (MΦs) in the subendothelial layer of arteries leading to foam cell and fatty streak formation. Many studies suggest that LDL that is modified by myeloperoxidase (MPO) is a key player in the development of atherosclerosis. MΦs can adopt a variety of functional phenotypes that include mainly the proinflammatory M1 and the anti-inflammatory M2 MΦ phenotypes which are both implicated in the process of atherogenesis. In fact, MΦs that reside in atherosclerostic lesions were shown to express a variety of phenotypes ranging between the M1- and M2 MΦ types. Recently, we pointed out the involvement of MPO oxidized-LDL (Mox-LDL) in increasing inflammation in MΦs by reducing their secretion of IL-10. Since little is known about Mox-LDL-mediated pro-atherosclerostic responses in MΦs, our study aimed at analyzing the in vitro effects of Mox-LDL at this level through making use of the well-established model of human THP-1-derived Mφs. Our results demonstrate that Mox-LDL has no effect on apoptosis, reactive oxygen species (ROS) generation and cell death in our cell model; yet, interestingly, our results show that Mox-LDL is significantly engulfed at a higher rate in the different MΦ subtypes supporting its key role in foam cell formation during the progression of the disease as well as previous data that were generated using another primary MΦ cell model of atherosclerosis.

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BACKGROUND: Atherosclerosis is a long-lasting inflammatory condition affecting the walls of arteries, marked by the buildup of fats, plaque formation, and vascular remodeling. Recent findings highlight the significance of cholesterol removal pathways in influencing atherosclerosis, yet the connection between cholesterol removal and regulation of macrophage inflammation remains poorly understood. RBAP could serve as an anti-inflammatory agent; however, its role in atherosclerosis and the mechanism behind it are still not well understood.
OBJECTIVE: The objective of this research is to explore how RBAP impacts cholesterol efflux, which is a considerable element in the advancement of atherosclerosis.
METHODS: An atherosclerosis mouse model was established by using an ApoE KO strain mouse on a high-fat diet (HFD) to assess the effects of RBAP, conducted either orally or through injection. Additionally, in vitro experiments were conducted where the induction of THP-1 cells was conducted for the differentiation towards macrophages, and along with mouse RAW264.7 cells, were challenged with ox-LDL to evaluate the impact of RBAP.
RESULTS: In this study, RBAP was found to reduce the production and downregulate TNF-α, IL-1β, and IL-6 levels and inhibited the activation of the TLR4/MyD88/NF-κB signaling in atherosclerosis model mice, as well as in ox-LDL-challenged THP-1 cells and mouse RAW264.7 macrophages. RBAP\'s effectiveness also improved the enhancement of reverse cholesterol transport (RCT) and cholesterol removal to HDL and apoA1 by increasing the activity of genes related to cholesterol removal PPARγ/LXRα/ABCA1/ABCG1, both in ApoE-/- mice and in THP-1 cells and mouse RAW264.7 macrophages. Notably, RBAP exerted similar effects on atherosclerosis model mice and macrophages to those of TAK-242, an inhibitor of the TLR4 signaling. When RBAP and TAK-242 were applied simultaneously, the improvement was not enhanced compared with either RBAP or TAK-242 treatment alone.
CONCLUSIONS: These findings suggest that RBAP, as a TLR4 inhibitor, has anti-atherosclerotic effects by improving inflammation and promoting cholesterol effection, indicating its therapeutic potential in intervening atherosclerosis.

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.

The accumulation of oxidized low-density lipoprotein (oxLDL) and its toxicity in the arterial wall have been implicated in atherosclerosis. This study aimed to investigate the mechanisms underlying the atheroprotective effect of bixin, a carotenoid obtained from the seeds of the tropical plant Bixa orellana, on Cu2+-induced LDL oxidation and oxLDL-mediated effects in J774A.1 macrophage cells. Bixin\'s effects were compared to those of lycopene, a carotenoid widely studied for its cardiovascular protective effects. LDL was isolated from human plasma, incubated with bixin or lycopene (positive control), and subjected to oxidation with CuSO4. Afterward, bixin or lycopene was incubated with J774A.1 macrophage cells and exposed to oxLDL. The levels of ROS, RNS, GSH, nitrite, mitochondrial function, and foam cell formation, as well as the expression of proteins related to the antioxidant and inflammatory status, were evaluated. The effect of bixin in inhibiting in vitro human-isolated LDL oxidation was more potent (5-6-fold) than that of lycopene. Bixin pretreatment reduced the atherogenic signaling triggered by oxLDL in the macrophages, namely the generation of reactive species, disturbance of nitric oxide homeostasis, mitochondrial dysfunction, and foam cell formation. The cytoprotective effects of bixin were accompanied by the upregulation of Nrf2 and the downregulation of the NF-kB pathways. Lycopene showed the same protective effect as bixin, except that it did not prevent mitochondrial dysfunction. The efficient performance of bixin makes it an ideal candidate for further trials as a new nutraceutical compound for the prevention of atherosclerosis.