As a chronic inflammatory condition, atherosclerosis features lipid deposition, leukocyte infiltration, and VSMC proliferation. An inflammatory environment in the plaque is decisively controlled by the activation of atheroma macrophages.

In early atherosclerosis, macrophage-derived NLRP3 inflammasome suggests beneficial effects on plaque stability, which are mediated by their involvement in inflammatory anti-injury reaction. NLRP3 inflammasome activation in late atherosclerosis causes macrophage death and a significant amount of lipid release, both of which increase plaque vulnerability [108, 109]. NLRP3 inflammasome activation can result from extracellular cholesterol crystal uptake, leading to lysosomal damage and the accumulation of cholesterol in the plasma membrane. Mice treated with NLRP3 inhibitors or NLRP3 genetic deletions suffer less atherosclerosis [110, 111].

The NLRP3 inflammasome is activated in two steps, first by priming and then by activating, which allows caspase 1 to release IL-1$\beta$ and IL-18 to mature forms. In fact, a variety of cell types can produce IL-6 when they are stimulated by IL-1$\beta$ or IL-18. A characteristic shift towards an acute phase pattern thus occurs in hepatocytes due to a change in protein synthesis pattern, which amplifies the inflammatory cascade inside the vessel [112].

Because the NLRP3 inflammasome sequelae are critical to the development of atherosclerosis by orchestrating the expression of inflammatory cytokines, it is likely that direct inhibition of inflammatory pathways through the NLRP3 inflammasome would be a promising therapy for atherosclerosis. At a molecular level, evidence suggests that autophagy, an intracellular degradation system that keeps cells in a state of homeostasis, down-regulates the activation of NLRP3 inflammasome [113]. A wide range of stimuli, including reactive oxygen species (ROS) from mitochondria, activate NLRP3 inflammasome, and autophagy inhibits their activation by the removal of damaged mitochondria [114]. Besides, The autophagic process inhibits inflammasome activity by degrading NLRP3 inflammasome via ubiquitination and modulating its activity. In fact, an autophagy defect in Atg 5${}^{\text{-/-}}$ mice is reflected in accelerated atherosclerosis and increased NLRP3 inflammasome activity. Furthermore, the phosphorylation of protein kinase A (PKA) is known to negatively regulate NLRP3 inflammasome. The G protein-coupled bile acid receptor (GP-BAR1) (also known as TGR5) activates PKA, resulting in the ubiquitination of NLRP3, which can be correlated with the phosphorylation of NLRP3. It has been shown that TGR5 can reduce the progression of atherosclerosis by promoting cholesterol efflux and reducing inflammatory reactions; however, their anti-inflammatory mechanisms remain to be fully clarified. The mechanisms by which NLRP3 inflammasome inhibits atherosclerosis also requires further studies [115, 116].

Results from the CANTOS and Cardiovascular Inflammation Reduction Trial suggest that the inhibition of the inflammasome through the CRP pathway lowers vascular risk [117]. Therefore, the NLRP3 inflammasome has attracted interest as a target in atherosclerosis due to its ability to generate both active forms of IL-1$\beta$ and IL-18. Nevertheless, further safety studies are needed for novel NLRP3-inflammasome inhibitors since such systemic inhibition of inflammatory pathways is likely to interact with immune homeostasis in ways that pose potential risks. Also, there is currently no data available to determine whether targeting IL-6, downstream of IL-1$\beta$ and IL-18, is a better option, which might theoretically mute inflammation with less impairment of host defenses [118].

Evidence is mounting that gut microflora plays a critical role in mucosal integrity and tolerance. Alterations in the gut microbiota may trigger an inflammatory state in the gut, which could lead to systemic inflammation. Dietary fibers are fermented in the gut microbiota, resulting in SCFAs, which are the main energy source for colonocytes, and they are presumably involved in colon epithelium renewal [119]. The decreased production of SCFAs that results from low dietary fiber consumption increases epithelial permeability by impairing epithelial metabolism. Therefore, either local or systemic inflammation may ensue as a result of the translocation of bacteria and LPS created by gut bacteria. Specifically, LPS binds to the TLR4 complex, along with the co-receptor CD14, which activates myeloid differentiation factor 88/NF-NF-$\kappa$B pathway, resulting in increased production of pro-inflammatory cytokines including IL-6, IL-1, IL-27, and TNF-$\alpha$, all of which are involved in atherosclerosis development [120]. Karlsson et al. [121] compared the gut microbiomes of atherosclerosis patients with control samples by functional characterization, and found that butyrate-acetoacetate CoA-transferase which originates from Clostridium sp. SS2/1 correlated negatively with high-sensitivity CRP in blood. In fact, butyrate is found to increase the generation of anti-inflammatory Treg cells and reduce inflammation in the colon [122].

Furthermore, the role of TMAO in accelerating atherosclerosis has recently received attention. After ingestion, bacteria residing in the gut convert L-carnitine and choline into trimethylamine, which is absorbed and converted by hepatic flavin monooxygenases to TMAO. The TMAO increases macrophage uptake of LDL-cholesterol and speeds foam cell formation by activating macrophage scavenger receptors CD36 and steroid receptor RNA activator 1, which explains its pro-atherogenic effects [123]. In the gut, TMAO precursors can be attenuated by administering poorly absorbed broad-spectrum antibiotics [123, 124]. TMAO further promotes atherosclerosis by activating the CD36-dependent MAPK/Janus kinases (JAKs) pathway and triggering the expression of VCAM-1, TNF-$\alpha$ and IL-1$\beta$ via the NF-$\kappa$B pathway [125, 126]. In addition, researchers have reported that TMAO causes hyper-reactivity in platelets, which can facilitate thrombosis, resulting in athero-thrombotic events [127].

In humans, circulating TMAO levels are associated with coronary artery disease burden and mortality in coronary artery disease patients in a dose-dependent fashion [128, 129]. The consumption of resveratrol can increase the ratio of Bacteroidetes to Firmicutes and the growth of Bacteroides, Lactobacillus, and Bifidobacterium, which have been shown to reduce levels of TMAO [130]. The possibility of modulating TMAO-producing bacteria to lower plasma TMAO levels sounds intriguing, but an effective treatment mode has not yet been identified.

Though TMAO appears to contribute to atherosclerosis in a variety of ways, the causal effect of TMAO on atherosclerosis is still being explored. In addition, even some studies have reported results contrary to the studies mentioning TMAO’s effect on atherosclerosis. In the report from Bäckhed laboratory, germ-free mice and conventionally raised Apolipoprotein E deficient (Apoe${}^{\mathit{\text{-/-}}}$) mice were fed either a chow or a Western type diet (WTD) containing standard low levels of choline (0.08%) or enriched in choline (1%–1.2%) [131]. As expected, in germ-free mice fed with chow diet or WTD, TMAO was barely detectable, and supplementation with choline did not increase plasma TMAO in germ-free mice, while conventionally raised mice showed an increase in plasma TMAO after supplementation with choline [131]. It is interesting to note that germ-free mice fed with chow diet had greater aortic root lesions than conventionally raised mice fed with chow diet; however, germ-free and conventionally raised mice fed with WTD had no difference in aortic lesions [131]. In mice fed WTD, the absence of microbiota had no effect on atherosclerosis [131]. In conventionally raised male mice, elevated plasma TMAO had no effect on atherosclerosis and associated lesions [131]. It was concluded that gut microbiota influences atherosclerosis in a dietary-dependent manner and is associated with plasma cholesterol levels [131]. Therefore, TMAO’s effects on atherosclerosis are not completely understood. Also, it is unclear how useful it is to measure TMAO levels in clinical scenarios, especially as there are no treatments to lower TMAO levels beyond lifestyle modifications in clinical practice.

Overall, the microbiota has been studied mostly through cross-sectional studies to date in relation to low-grade inflammation and cardiovascular disease. We need to conduct further prospective studies to confirm these findings. With respect to how the gut microbiota can be regulated and how to break the possible feedback loop between gut inflammation and systemic inflammation, a strategy that employs prebiotics, probiotics, and natural products to promote beneficial changes in the intestinal flora and to improve epithelial integrity seems promising.

The major atherosclerotic plaque cells present in the fibrous cap and around the necrotic core (e.g., ECs, VSMCs, macrophages) undergo autophagy. The role of EC autophagy in atherosclerosis progression is still debated. One study shows that oxidized LDL (ox-LDL) increases the von Willebrand factor and P-selectin secretion in ECs, indicating that this may be a mechanism through which ox-LDL inhibits the sirtuin-1/forkhead box transcription factor O1 pathway and increases autophagic flux in ECs. Hence, reducing arterial thrombosis and atherosclerosis might be possible by increasing autophagic flux [132]. Endothelial autophagy also inhibits vascular inflammation through the down-regulation of ICAM-1, VCAM-1, E-selectin, and NF-$\kappa$B signaling [133]. Induction of autophagy in TNF-stimulated human umbilical vein ECs (HUVECs) reduces the expression of inflammatory cytokines via activation of the PKA/adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK)/SIRT1 pathway [134]. Other studies, however, have shown that EC autophagy promotes atherosclerosis. Autophagic death induced by excessive autophagy in ECs may destroy the stability of atherosclerotic plaques [135]. In addition, stress-induced overactivated autophagy increases apoptosis in ECs, while recombinant thrombomodulin treatment can inhibit EC apoptosis by modulating rapamycin (mTOR)-dependent autophagy [136].

As for VSMC autophagy in atherogenesis, a study by Pi S et al. [137] demonstrated that the activation of the P2RY12 receptor triggered mTOR through the phosphatidylinositol 3-kinase/Akt pathway, resulting in the inhibition of autophagy in advanced atherosclerosis and reduced cholesterol outflow. However, miR-223 inhibited the formation of foam cells via inducing the autophagy of VSMCs [138]. According to another study, ox-LDL induced atherogenesis is inhibited by AMPK/mTOR signaling, which increases autophagy [139]. The knockdown of sterol regulatory element-binding cleavage-activating proteins in VSMCs of ApoE${}^{\mathit{\text{-/-}}}$ mice reduces lipid accumulation and oxidative stress as well as stimulates VSMC autophagy via ROS/AMPK pathways [140]. Nevertheless, in VSMCs, tissue-specific deletion of Atg7 causes accumulation of SQSTM1/p62 and accelerates stress-induced premature senescence [141]. In this way, senescent VSMCs develop an inflammatory, senescence-associated secretory phenotype driven by IL-1$\alpha$ that may contribute to atherosclerosis [142]. Similarly, VSMCs that undergo excessive autophagy destabilize plaques by reducing collagen synthesis, resulting in a thinner fibrous cap [143].

The autophagic process is closely linked to the formation and development of atherosclerotic plaques composed of macrophage-derived foam cells. By selectively inhibiting the PI3K/Akt/mTOR pathway, autophagy is activated in macrophages, improving atherosclerotic plaque stability [144]. By degrading the NLRP3 and ASC subunit of the inflammasome, macrophage autophagy is shown to inhibit inflammation in ox-LDL-induced foam cells [145]. By contrast, atherosclerosis occurs and is exacerbated by impaired or defective autophagy in macrophages. Defects in macrophage ATG5 promote apoptosis and oxidative stress in macrophages, and worsen lesional efferocytosis in advanced atherosclerosis [146]. Therefore, activating macrophage autophagy may offer new therapeutic approaches to treat atherosclerosis.

The above results show different autophagy levels in different cells, which can be manifested as defective, basal, mild or over-induced autophagy, accompanies the entire process of atherogenesis. A growing body of evidence suggests that while the basic or moderate form of autophagy suppresses inflammation, increases cell survival, and protects against the progression of atherosclerotic plaques, the impaired or excessive form promotes inflammation, and accelerates cell death, and apoptosis, further exacerbating plaque instability and rupture. As such, targeting atherosclerotic plaque cell autophagy could be a viable approach for atherosclerosis treatment, which aims to control autophagy without causing harmful consequences.

Several atherogenic processes may be influenced by lncRNAs, which are defined as non-protein-coding transcripts longer than 200 nucleotides. By regulating autophagic flux, lncRNAs act as molecular switches that regulate lipid metabolism and the inflammatory response in the vasculature despite their poor conservation between species. This opened up the field for the investigation into relationships among lncRNAs, autophagy and atherosclerosis.

For example, through binding mixed lineage kinase domain-like protein (MLKL) promoter, lncRNA FA2H-2 regulates autophagy and inflammation in atherosclerosis. After the suppression of MLKL expression in SMCs and ECs in response to ox-LDL, autophagic flux is enhanced and inflammation is diminished [147]. The reduction of FA2H-2 expression in VSMCs and ECs leads to a significant increase in the expression of MLKL, suppresses autophagic flux, and induces the expression of IL-1$\beta$, IL-6, and IL-8, and TNF-$\alpha$ [147]. FA2H-2 promotes autophagy in VSMCs and ECs, thus reducing inflammation by acting as an atheroprotector.

MALAT1 is another interesting lncRNA under investigation. Recently, a study demonstrated that MALAT1 possesses anti-inflammatory properties in part through its binding to miR-503 [148]. Mechanistically, atherosclerotic plaque formation is mitigated by MALT1 by blocking the adhesion of myeloid cells to ECs and reducing pro-inflammatory cytokine production [148].

Overall, lncRNAs, as a regulator, may have cell-type-specific expression patterns, and are appealing pharmacological targets. In order to develop therapeutic strategies targeting lncRNAs for atherosclerosis, it is essential to unearth how lncRNA-mediated autophagy is regulated in atherosclerotic plaque cells.

The present study also identified EVs, especially exosomes as another emerging topic in the field. The extracellular vesicles called exosomes are nanosized with a diameter of 30 to 150 nm and contain the RNA, DNA, proteins, and lipids from the donor cells [149]. Exosomes can interfere with the function of ECs, VSMCs, and macrophages, causing either pro-atherosclerosis or anti-atherosclerosis, depending on the donor cells’ condition.

For example, exosomes secreted by ox-LDL-stimulated THP-1 monocyte ferrying lncRNA LIPCAR, miR-106a-3p, and GAS5 are reported to promote atherosclerosis by altering the phenotypes of ECs and VSMCs [150, 151, 152]. By contrast, exosomal miRNAs derived from mesenchymal stem cells, bone marrow-derived macrophages, as well as platelets may exert anti-atherosclerotic effects [153, 154, 155]. In addition, although low levels of lncRNAs are present in exosomes, increasing data suggest that exosomal lncRNAs are new clues to the pathogenesis of ASCVD and more attention is needed to help clarify the possible relationships among exosomes, exosomal lncRNAs and miRNAs [156].

The Nrf-2 protein encoded by the NFE2L2 gene has been closely linked with atherosclerosis, yet its role seems antagonistic, preventing as well as promoting its development. Depletion of Nrf2 in bone marrow-derived cells is shown to attenuate the formation of atherosclerotic plaques, whose mechanism probably implicates the reduction in pro-inflammatory M1 macrophage [157]. The Nrf2-mediated role in potentiating atherosclerosis also involves NLRP3 inflammasome activation, decreased uptake of acetylated LDL, and up-regulated expression of CD36 scavenger receptor in macrophages [158, 159, 160, 161].

By contrast, the activation of Nrf2/HO-1 signaling is shown to decrease gene expressions of inflammatory factors such as TNF-$\alpha$, IL-1$\beta$ and IL-6 in LPS-stimulated RAW264.7 cells, which highlights the anti-inflammatory activity of Nrf2 [162]. Hu Q et al. [163] has found that dihydromyricetin pre-treatment suppresses pyroptotic cell death and reduces IL-1$\beta$ release by the activation of the Nrf2 signaling to inhibit NLRP3 inflammasome activation in palmitic acid-induced HUVECs. In addition, Nrf2/HO-1 signaling is a popularly researched pathway through whose activation natural extracts protect ECs from injury in atherosclerosis [164].

Thus, in vitro and in vivo experiments with Nrf2 in atherosclerosis are, however, ambiguous and heavily dependent on the atherosclerotic lesion stage or animal model. Also, further research is needed to better define its contribution to human atherosclerosis.

The reasons for these foregoing topics being research hotspots are multifaceted either because these targets began to emerge in the most recent years or because the lack of suitable methodologies in earlier studies have made it challenging to investigate their involvement in ASCVD.

As a result of advances in biochemistry, cell biology, and genetic engineering, experimental atherosclerosis has experienced a flurry of activity, leading to thousands of experimental papers that shed light on diverse aspects of inflammation in the regulation of ASCVD. In spite of these impressive experimental results, a gap remained between the lab and the clinic.

In experimental aspect, despite the availability of ApoE${}^{\mathrm{\text{-/-}}}$ and the LDL receptor-deficient (LDLr${}^{\mathrm{\text{-/-}}}$) mice, studies were conducted using mice to investigate the effects of the potent proinflammatory cytokines, IL-1$\beta$, and IL-18 on atherosclerosis in the early 2000s [165, 166]. It was not until nearly ten years later that Duewell P et al. [110] published their landmark study that showed atherosclerotic lesions in ApoE deficient mice on a high-fat diet contained cholesterol crystals that increased as the disease progressed. In particular, these crystals were observed to co-localize with NLRP3 inflammasomes and promoted the release of IL-1$\beta$ [110]. Further, it was shown that LDLr${}^{\mathit{\text{-/-}}}$ mice that received bone marrow transplants with NLRP3-deficient, ASC-deficient, and IL-1$\alpha$/$\beta$-deficient bone marrow showed markedly reduced atherosclerosis [110]. Due to the cross-talk between inflammation and lipid metabolism that the NLRP3 inflammasome mediates, intensive studies have been been spotlighted on the role of the NLRP3 inflammasome in atherogenesis in mouse models, including its activation in ECs, immune cells (such as monocytes, macrophages and dendritic cells), and SMCs as well as its mechanism of action in atherosclerotic disease since then. In spite of the proliferation of NLRP3 inflammasome research topics in basic science, its clinical application seems stagnant as few biomarkers of inflammation have moved from the lab to the clinical setting.

With regard to clinical research, also in the early 2000s, lipid-lowering trials demonstrated that statin therapy reduced the level of circulating hsCRP, which confirmed at least partially the interaction between inflammation and lipid metabolism [167, 168, 169]. It was identified in landmark statin trials that patients who demonstrated reductions in LDL-cholesterol to less than 70 mg/dL as well as a reduction in hsCRP to less than 2 mg/L had a greater cardiovascular benefit than those with only a significant reduction in LDL-cholesterol levels [170, 171, 172]. A consequence of this has led to the concept of residual inflammatory risk, defined as persistently elevated hsCRP despite adequate atherogenic lipid lowering, a phenomenon seen in 30%–40% of all statin trial participants [173].

Observational studies have shown strong associations between CRP levels, coronary atherosclerosis, and vascular risk [174, 175]. In spite of the fact that CRP does not directly contribute to atherothrombosis [176], its application as a biomarker of inflammation has begun to bridge the gap between experimental and clinical findings. In the decade that have followed the initial investigation of CRP in cardiovascular disease, dozens of studies have accumulated evidence that strongly associates inflammation as measured by CRP with a number of outcomes associated with a range of cardiovascular outcomes [177]. As far as other biomarkers of inflammation and oxidative stress are concerned, none have proven to be as reproducible and clinically practical as CRP measured with the highly sensitive method [178].

Most importantly, CRP integrates inflammatory signals generated by a variety of sources. As previously mentioned, while statin therapy, such as rosuvastatin, may promote some of its clinical benefit through direct anti-inflammatory effects, because of concomitant reduction in LDL [179], it is not possible to determine rigorously the extent to which this agent’s anti-inflammatory effects contribute to the clinical benefit observed, independent of LDL lowering.

With the identification the canonical pathway that links the NLRP3 inflammasome with IL-1/IL-6/CRP [180], targeting the NLRP3 inflammasome may provide therapeutic benefits in ASCVD, especially in light of the results of clinical trials such as CANTOS, which demonstrated that inhibiting inhibiting IL-1$\beta$, a main product of NLRP3 inflammasome activation, diminished clinical endpoints in patients with atherosclerotic disease independent of lipid level lowering, accompanied by a reduction in IL-6 and CRP [66, 181]. Also, methotrexate failed to inhibit IL-6/IL-1$\beta$ pathway and acted via the inhibition of aminoimidazole-4-carboximaide ribonucleotide, resulting in subsequent elevations in adenosine levels, and no reduction in cardiovascular endpoints [182], further suggesting inflammation that leads to atherogenesis is pathway-specific rather than generalized. For all these reasons, it appears the NLRP3 inflammasome is linked to ASCVD and understanding the mechanisms regulating NLRP3 inflammasome activation and subsequent signaling is vital to gain insight into potential therapeutic strategies to prevent ASCVD, which makes NLRP3 inflammasome a research hotspot in this field.

With regard to the research on gut microbiota, the atherosclerotic plaque has been found to contain a number of bacteria, including Streptococcus, Pseudomonas, Klebsiella, Veillonella spp., and Chlamydia pneumoniae, making it a microbial environment on itself [183, 184, 185]. Most studies, however, failed to link plaque microbiota composition with outcomes such as plaque vulnerability, rupture, or cardiovascular events [186]. In addition, antibiotic treatment as secondary prevention, which seeks to eliminate plaque microbiota, did not result in a reduced incidence of cardiovascular events [187]. As a result, these studies failed to provide evidence in support of the causal role of direct vessel wall infection in plaque formation. In contrast, the hypothesis that distant infections can elicit an auto-immune inflammatory response through molecular mimicry appears to be more plausible [188].

Since the development of 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing, we have therefore received a greater understanding of the role of gut microbiota in ASCVD [189]. Specifically, metagenomic sequencing allows not only species-level resolution of compositional data, but also enables the assessment of differences in gut microbiota functionality, since compositional differences do not always translate into functional differences. Human cross-sectional studies found that patients with symptomatic atherosclerosis have higher abundances of Collinsella genus, Enterobacteriaceae, Streptococcaceae, and Klebsiellaspecies, as well as lower abundances of SCFA-producing bacteria Eubacterium, Roseburia, and Ruminococcaceae spp. as compared with healthy controls in the gut microbiota [121, 190, 191]. In addition, with the introduction of metabolomics approach, it has been shown that plasma levels of TMAO and its precursors, choline and betaine has been shown to predict the risk of cardiovascular disease [124].

The advent of culture-independent sequencing technologies and omics led to a huge amount of associative data, but these data are not robust to causal investigation, despite their usefulness. Further, in animal studies, fecal microbiota transplantation (FMT) provides causal evidence that gut microbiota composition is associated with atherosclerosis. For instance, mice transplanted with a microbiota composition that was more pro-inflammatory from Caspase1${}^{\mathit{\text{-/-}}}$ mice had 29% larger plaques than those in the control group [192]. In this regard, gut microbes could indirectly contribute to atherosclerosis by producing pro-atherogenic metabolites.

Overall, the spotlight on the role of gut microbiota and metabolites such as TMAO in atherosclerosis benefits both from the advancement in sequencing technologies, bioinformatics tools, and omics technologies, and the efforts to determine whether microbial changes are driving disease states or rather being driven by them through the introduction of emerging methodological approach (e.g., FMT).

Over 50 years ago, autophagy was identified as a mechanism that sequesters and degrades cytosolic components via the lysosome pathway; however, the role of autophagy in atherosclerotic lesions remains unappreciated due to incompletely characterized ultrastructural features. The first hint for its contribution in atherosclerosis came from the paper by Perrotta I [193] in 2013, in which transmission electron microscopy revealed autophagy in all primary cell types (i.e., macrophages, VSMCs, and ECs) in human atherosclerotic plaques. Since then, a growing number of studies have provided further evidence that autophagy occurs in atherosclerosis with the expression of autophagy marker, such as microtubule-associated protein light chain 3-II and microtubule-associated protein 1 light chain 3 detected in various cells in unstable atherosclerotic plaques [194].

Therefore, the cell-specific contributions of autophagy to atherosclerosis initiation, progression, and regression were investigated. In addition, with the discovery of other forms of regulated cell death such as pyroptosis and ferroptosis in recent years [195], their intricate overlapping effects and interactions have further increased interest in autophagy as a hot topic in atherosclerosis research.

The role of exosomes in cardiovascular diseases has, as evidenced by a bibliometric analysis, emerged as a “star target” only in the last decade; especially in the past five years, the area has gained significant momentum [196]. Exosomes are crucial mediators of intercellular communication during atherosclerosis development, as described above; a major obstacle to the reaseach on exosomes is the isolation methods, since obtaining high-purity exosomes is crucial for further research.

While there is currently no standardized method for the isolation of exosomes, many techniques have been developed based on the biochemical and physicochemical characteristics of exosomes, including differential ultracentrifugation, immunoaffinity capture, polymer-based precipitation, ultrafiltration, and size exclusion chromatography, each of which has its own advantages and disadvantages [197, 198]. These significant methodological advances have resulted in further insights into the role that intercellular communication plays in ASCVD pathogenesis, thereby leading to exosomes as an emerging mediator of atherogenesis. It is expected that with the introduction of promising exosome isolation methods very recently, such as ExoTIC, acoustofluidic platform, and alternating current electrokinetic microarray chip devices [199, 200, 201], further interest will be fueled in exosomal research, allowing them to be used in clinical settings for diagnostic or therapeutic purposes.

Non-coding RNAs (ncRNAs) were initially regarded as transcriptional noise or residual waste generated during the processing of RNA. Nevertheless, research interest expanded to miRNA in early 2000, with subsequent studies showing that miRNAs play a significant role in physiological processes and pathological outcomes. In fact, the first evidence for a putative role of ncRNA in vascular disease came from genome-wide association studies that identified the most significantly associated locus with coronary artery disease on the human chromosome 9p21 [202]. The region is adjacent to INK4 locus that encodes a lncRNA named ANRIL, also known as CDKN2BAS [203]. As opposed to miRNAs, whose biosynthesis and biological activities are well explored, lncRNAs are more heterogeneous and difficult to characterize. It is not easy to infer the specific function of lncRNAs from their sequence or structure, unlike microRNAs or proteins.

However, the field of lncRNAs has benefited from the advance in genomic technologies, including the availability of fast and cost-effective sequencing technologies and computational resources, with a new class of lncRNAs discovered and annotated every year. In this regard, lncRNAs have been shown to be expressed in major types of cells in atherosclerotic lesions and are implicated in several atherogenic processes [204].

Furthermore, recent studies have concentrated on the characterization of molecules within exosomes, as well as their use as diagnostic agents. For example, exosomal lncRNA HIF1A-AS1 was found by Wang Y et al. [205] to be a potential biomarker for atherosclerosis. In this way, by rapidly advancing technologies and protocols for isolating, purifying, and detecting exosomes, in addition to rapidly developing genomic tools, exosomal lncRNAs are also positioned to undergo clinical translation.

Since Nrf2 was first cloned in 1994, the field of Nrf2 is relatively young. It was in the mid- to late 1990s, when the first Nrf2${}^{\mathrm{\text{-/-}}}$ mouse was generated [206], that many of the studies were conducted on molecular interactions that drive Nrf2 signaling. Moreover, the Nrf2${}^{\mathit{\text{-/-}}}$ mouse provided insight into Nrf2’s role in chemoprevention as oltipraz, a dithiolethione, lost its chemoprotective effects in mice deficient in Nrf2 [207]. Upon revealing that Nrf2 plays a chemoprotective role, the focus shifted toward identifying methods for activating the Nrf2 pathway.

As recently as 2006, however, the discovery that KEAP1 mutations that leads to chronically elevated levels of Nrf2 was found in non-small lung cell carcinomas presented the first evidence that Nrf2 may contribute to cancer progression and chemoresistance [208], which was later referred to as the “dark side” of Nrf2. Despite Nrf2’s known benefits, new research has revealed the previously unappreciated complexity of the Nrf2 signaling network, growing evidence that careful regulation of this pathway is crucial to disease prevention. In the field of atherosclerosis research, the same applies. Studies have demonstrated that Nrf2 plays a dual role in atherosclerosis, as previously described. The paradoxical role of Nrf2 in atherosclerosis thereby spurs further research into its role in the various stages of plaque progression before it is considered a new therapeutic target due to the relative lack of understanding regarding its complex and diverse mechanisms.