Targeting Foam Cell Formation in Atherosclerosis: Therapeutic Potential of Natural Products

1. CD36

CD36 (also known as fatty acid translocase, FAT) belongs to class B of the SR family, representing 88 kDa heavily glycosylated transmembrane platelet glycoprotein III b/IV (Yu et al., 2013). It was first identified in 1993 as a macrophage receptor that binds (with high affinity), internalizes, and degrades oxLDL (Endemann et al., 1993). CD36 has an extracellular domain, two cytoplasmic domains, and two transmembrane domains. CD36 is ubiquitously expressed in multiple cell types, including monocytes/macrophages, endothelial cells, platelets, and many others (Yu et al., 2013). In addition to regulating modified LDL binding, CD36 has other functions as well, such as involvement in inflammatory processes, lipid metabolism, fatty acid transport, and immunity (Febbraio et al., 2001). Another important function of CD36 is to modulate the migration of macrophages upon oxLDL stimulation, which may contribute to macrophage trapping/retention in arterial lesions (Park, 2014). CD36 can also be released to the circulation in patients with atherosclerosis, which leads to the formation of soluble CD36 (Handberg et al., 2008). Thus, soluble CD36 is a promising biomarker of plaque instability and symptomatic carotid atherosclerosis, as well as associated with carotid intima-media thickness (Handberg et al., 2008; Jiang et al., 2017b).

The expression of CD36 in macrophages is upregulated by several proatherogenic stimuli, such as oxLDL (Endemann et al., 1993), palmitate (Kim et al., 2017), and dysfunctional HDL from coronary artery disease (CAD) patients (Sini et al., 2017). The CD36 gene transcription is regulated by peroxisome proliferator-activated receptor-γ (PPARγ) (Nagy et al., 1998; Tontonoz et al., 1998), nuclear erythroid-related factor 2 (Nrf2) (Ishii et al., 2004; Olagnier et al., 2011), as well as by signal transducer and activator of transcription 1 (STAT1) (Kotla et al., 2017) pathways. In addition, CD36 expression can also be regulated by retinol-binding protein 4 (Liu et al., 2017b), protein kinase Cθ (PKCθ)/activating transcription factor 2 (Raghavan et al., 2018), epidermal growth factor receptor (Zeboudj et al., 2018), trimethylamine N-oxide (Geng et al., 2018), NACHT, LRR, and PYD domains-containing protein 3 inflammasome (Chen et al., 2018b), transient receptor potential vanilloid 4 (TRPV4) (Goswami et al., 2017), cellular communication network factor 3 (Shi et al., 2017), melanocortin 1 receptor (Rinne et al., 2017), cluster of differentiation 146 (CD146) (Luo et al., 2017), triggering receptor expressed on myeloid cells (Joffre et al., 2016), cyclophilin A (Ramachandran et al., 2016), and many others. Furthermore, CD36-mediated foam cell formation and atherosclerosis were negatively regulated by C1q/tumor necrosis factor-related protein 13 via autophagy/lysosome-dependent degradation of CD36 (Wang et al., 2019a).

Recently, noncoding RNAs (ncRNA), such as miRNAs and lncRNAs, were proposed as a new layer of gene regulation in cardiovascular biology. A few miRNAs have been shown to regulate the CD36 expression, thus influencing foam cell formation and lipid accumulation in macrophages (Dai et al., 2016), including miRNA-155 (Huang et al., 2010). The CD36 expression is also regulated by lncRNAs. For example, lncRNA RP5-833A20.1 interacted with miRNA-382-5p to suppress nuclear factor I A (NFIA), thus increasing the expression of nuclear transcription factor-κB (NF-κB), SR-A, and CD36, leading to increased cholesterol accumulation and higher inflammatory response (Hu et al., 2015). lncRNAs nuclear paraspeckle assembly transcript 1 (Neat1), which has two isoforms, Neat1_1 (shorter) and Neat1_2 (longer), regulate the formation of paraspeckles (subnuclear structures). Two recent studies have shown that Neat1 is an oxLDL inducible lncRNA that regulates the macrophage inflammation, oxidative stress, apoptosis, and foam cell formation (Chen et al., 2018a; Huang-Fu et al., 2018). Similarly, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a macrophage-enriched and oxLDL inducible lncRNA, also involves in regulation of oxLDL-induced CD36 expression and oxLDL uptake through activation of β-catenin (a transcriptional factor for CD36) (Huangfu et al., 2018). Despite reduced uptake of oxLDL by MALAT1 deficiency, heterozygous deletion of MALAT1 aggravated atherosclerosis in ApoE-deficient (ApoE^−/−) mice by inducing massive immune system dysregulation (Gast et al., 2019). Gain- and loss-of-function assays of homeobox protein transcription antisense RNA (HOTAIR) in oxLDL-stimulated macrophages suggest that HOTAIR controlled CD36 expression, oxLDL uptake, inflammation [interleukin-6 (IL-6), IL-1β, cyclooxygenase 2 (COX2), and tumor necrosis factor-α (TNF-α)], and macrophage-derived foam cell formation via miRNA-330-5p (Liu et al., 2019). Therefore, ncRNAs present potential targets to fine-tune macrophage function within atherosclerotic lesions. For a detailed review of ncRNAs in regulating SRs, we refer to a recent review (Dai et al., 2016).

Studies performed using CD36^−/− mice to dissect the role of CD36 in atherosclerosis have yield controversial results (Collot-Teixeira et al., 2007). For example, targeted disruption of CD36 reduced the oxLDL uptake, foam cell formation, and atherosclerotic plaques in ApoE^−/− mice (Febbraio et al., 2000). This was supported by an independent study using shRNA-mediated gene silencing of CD36, which showed that silencing of CD36 in bone marrow-derived cells decreased atherosclerotic plaques (Mäkinen et al., 2010). Another study showed that CD36^−/−-deficient male ApoE^−/− mice, but not female mice, showed reduced atherosclerotic plaque area after feeding normal diet and Western-type diet; however, this was unexpectedly related to increased aortic sinus lesion areas (Moore et al., 2005). In contrast, a second study observed that CD36 deficiency reduced atherosclerosis in both sexes of ApoE^−/− mice (Kuchibhotla et al., 2008). A third study observed increased atherosclerosis only in male CD36^−/− and ApoE^−/− mice fed a Western-type diet, albeit with reduced foam cell formation in vitro; however, simultaneous deletion of both CD36 and SR-A significantly reduced atherosclerosis as well as plaque necrosis in ApoE^−/− mice without affecting foam cell formation (Manning-Tobin et al., 2009). These discrepancies in phenotypic observations suggest that CD36 in other cell types (such as endothelial cells, VSMCs, monocytes, and dendritic cells) may have lipid uptake-independent functions, and the possible reciprocal interaction network among SRs may be complicatedly regulating lipid metabolism and atherosclerosis (Maguire et al., 2019).

2. Macrophage Scavenger Receptor 1

SR-A (also known as CD204, or macrophage scavenger receptor 1) is a 77-kDa glycoprotein belonging to class A of the SR family. SR-A is the first identified and cloned SR in macrophages (Kodama et al., 1990). Alternative splicing of SR-A leads to the generation of several variants, such as SR-A1/2/3 (human SR-A) and SR-A1/2 (mouse SR-A). SR-A gene is located on chromosome 8 (8p22). SR-A protein has a cysteine-rich C-terminal domain, an extracellular domain, and a collagen-like domain (McLaren et al., 2011a). SR-A1/2s are the common functional receptors that mediate the endocytosis of modified LDLs, such as acetylated LDL (acLDL) (Terpstra et al., 1997). The expression of SR-A was upregulated by multiple proatherogenic stimuli [such as acLDL (Terpstra et al., 1997), oxLDL (Terpstra et al., 1997), TNF-α (Hashizume and Mihara, 2012), IL-6 (Hashizume and Mihara, 2012), high glucose (Fukuhara-Takaki et al., 2005), etc.] and downregulated by many plant-derived phytochemicals (Yu et al., 2013). SR-A1 gene transcription is primarily regulated by PU.1 (Horvai et al., 1995), NF-κB (Hashizume and Mihara, 2012), activating protein-1 (AP-1) (Mietus-Snyder et al., 1998), and CCAAT/enhancer binding protein (C/EBP) (Mietus-Snyder et al., 1998). Several miRNAs were shown to regulate the SR-A expression, such as miRNA-29a (Dai et al., 2016). SR-A1 expression can be upregulated through Orai1 store-operated calcium channel (Liang et al., 2016), chloride channel ClC₃ (Tao et al., 2015b), and endophilin-A2 (Huang et al., 2016a).

Earlier studies in the 1990s showed that deletion of SR-A can reduce atherosclerosis development in both LDL receptor-deficient (LDLR^−/−) mice (Sakaguchi et al., 1998) and ApoE^−/− mice (Suzuki et al., 1997). The pathologic role of SR-A is supported by its silencing in LDLR^−/−ApoB100 mice. However, simultaneous depletion of SR-A and CD36 did not reduce atherosclerosis, due to reciprocal regulation of both receptors (Mäkinen et al., 2010). Follow up studies suggested the more complex role of SR-A in atherosclerosis. For example, deficiency of either SR-A or CD36 alone had no impact on atherosclerotic development in ApoE^−/− mice, although it attenuated foam cell formation (Moore et al., 2005), but SR-A and CD36 double knock-out reduced plaque complexity without significant impact on macrophage-derived foam cell formation. Also, SR-A transgenic in either LDLR^−/− mice or ApoE-3 Leiden mice decreased the atherosclerosis development despite increased foam cell formation in SR-A overexpressed macrophages (De Winther et al., 2000). This discrepancy may be attributed to several factors, including different diet, time of feeding, and different atherosusceptible mouse strain. To avoid the systemic effects caused by global knockout, macrophage-specific overexpression of SR-A was done and unexpectedly led to attenuated atherosclerosis development in LDLR^−/− mice (Whitman et al., 2002). Similarly, overexpressing SR-A in LDLR^−/− mice (via bone marrow transplantation) does not affect atherosclerotic lesion development despite increased SR activity in vitro (Herijgers et al., 2000). In addition to regulating lipid uptake, SR-A also has other biologic functions, such as regulation of vascular inflammation, host defense, innate immunity, cell fate determination, and ischemic injury, highlighting that SR-A could be a double-edged sword implicated in cardiovascular health as well as in multiple cardiovascular diseases (CVD) (Kelley et al., 2014; Ben et al., 2015).

3. Lectin-like Oxidized Low-density Lipoprotein Receptor-1

LOX-1 (lectin-like oxLDL receptor-1) belongs to the Class E of scavenger receptors located at chromosome 12 (12p13.2). LOX-1 is encoded by the gene-oxLDL receptor 1 (OLR1). LOX-1 was identified in 1997 as a major SR in endothelial cells that binds, internalizes, and degrades oxLDL (Sawamura et al., 1997). Genetic variants of LOX-1 were associated with increased risk of CVD (Chen et al., 2003; Wang et al., 2010c; Morini et al., 2016). The human LOX-1 gene has six exons and five introns, encoding LOX-1 protein, which has 273 amino acid residues. LOX-1 is composed of a C-type lectin-like domain, an extracellular domain, a transmembrane domain, and an N-terminal cytoplasmic domain (Dunn et al., 2008; Ogura et al., 2009). The C-type lectin domain is critical for oxLDL binding. In terms of expression, LOX-1 is ubiquitously expressed in vascular cells, such as endothelial cells (Sawamura et al., 1997), macrophages (Draude et al., 1999), VSMCs (Zhang et al., 2017), cardiomyocytes (Schlüter et al., 2017), and fibroblasts (Liu et al., 2016a). The ubiquitous expression pattern of LOX-1 in cardiovascular cells implicates its important role in regulating cardiovascular pathophysiology. In patients with advanced atherosclerotic plaques, LOX-1 protein expression was markedly increased in lesional macrophages and VSMCs (Kataoka et al., 1999). In addition, LOX-1 is cleaved to form soluble LOX-1, which serves as a diagnostic and prognostic biomarker for CAD (Tian et al., 2019a).

To date, oxLDL is the most common and well-characterized ligand for LOX-1 (Yoshimoto et al., 2011). Other ligands of LOX-1 include carbamylated LDL (Speer et al., 2014; Holy et al., 2016), glycoxidized LDL (Shiu et al., 2009), electronegative LDL fraction L5 (Wang et al., 2018g), and advanced glycation end-products (Jono et al., 2002). Expression of LOX-1 under basal unstimulated conditions is very low, but LOX-1 expression can be significantly upregulated by several atheroprone stimuli, such as disturbed blood flow (Lee et al., 2018), lipopolysaccharide (LPS) (Zhao et al., 2014), TNF-α (Kume et al., 2000), and high glucose (Li et al., 2003). Dysfunctional HDL from patients with CAD can also cause endothelial dysfunction by reducing endothelial nitric oxide (NO) synthase (eNOS)-dependent NO production, anti-inflammatory responses, and endothelial repair capacity via LOX-1 activation (Besler et al., 2011; Xu et al., 2013b). Similarly, dysfunction of HDL could probably lead to LOX-1-mediated uptake of modified LDL and result in foam cell formation.

LOX-1 gene transcription can be regulated by several transcriptional factors, including NF-κB, AP-1, and POU-domain transcription factor (Oct-1) in a context-dependent manner (Hermonat et al., 2011). LOX-1 gene expression can be epigenetically mediated by several miRNAs. For example, silencing of miRNA-155 promoted oxLDL-elicited lipid uptake by increasing the expression of LOX-1 (Huang et al., 2010). A recent miRNA transcriptional profiling assay showed that LOX-1 is the target of miRNA-30c-1-3p and miRNA-28a-5p in oxLDL-stimulated RAW264.7 macrophages (Li et al., 2018b). Sirtuin 1 (Sirt1), a class III histone deacetylase, can downregulate LOX-1 expression and LOX-1-mediated foam cell formation, as well as attenuate development of atherosclerosis by deacetylating in macrophages (Stein and Matter, 2011). It remains to be investigated whether there are lncRNAs that can regulate LOX-1 expression and LOX-1-mediated foam cell formation.

LOX-1 transgene accelerates endothelial dysfunction, aortic inflammation, intramyocardial vasculopathy, and atherosclerotic development in ApoE^−/− mice (Inoue et al., 2005). Experiments using endothelial cell-specific overexpression of LOX-1 obtained similar phenotype in either ApoE^−/− or LDLR^−/− mouse background by impairing endothelial function (White et al., 2011; Hofmann et al., 2017). In contrast, LOX-1^−/− and LDLR^−/− mice show reduced collagen deposition, oxidative stress, and developed fewer atherosclerotic plaques (Mehta et al., 2007; Hu et al., 2008). These series of gain- and loss-of-function studies provide the “proof-of-concept” that LOX-1 is a proatherogenic SR and can serve as a promising therapeutic target for atherosclerosis. The molecular mechanisms of LOX-1 in promoting atherosclerosis include induction of endothelial dysfunction (injury, apoptosis, impaired NO production, and endothelial-mesenchymal transition), the proliferation/migration of VSMCs, macrophage-derived foam cell formation, and platelet activation (Tian et al., 2019a).

For a detailed understanding of the functions and pharmacological modifiers of LOX-1 in atherosclerosis, we refer the readers to a recently published review (Tian et al., 2019a). More studies are needed to understand the specific role of LOX-1 in macrophage foam cell formation and atherosclerosis (by macrophage-specific knockout mice and/or transgenic mice) (Maguire et al., 2019). Also, LOX-1 was expressed in VSMCs in mice and human atherosclerotic plaques, the potential role of LOX-1 in lipid uptake and foam cell formation in VSMCs remains to be investigated.

4. Others

Other SRs that mediate lipid uptake include MARCO (Elomaa et al., 1998), SR expressed in endothelial cells (Adachi et al., 1997), and CXCL16 (Shimaoka et al., 2000). For example, CXCL16 was a SR highly expressed in macrophages (Shimaoka et al., 2000). Knockdown experiments have demonstrated that CXCL16 was important for foam cell formation in human macrophages (Zhang et al., 2008). It is plausible that these less common SRs also contribute to the uptake of modified LDL to certain extent or under specific disease conditions (McLaren et al., 2011a). Further studies are warranted to assess the contribution of these SRs to foam cell formation and atherosclerosis in genetically manipulated mice.

1. Acetyl-CoA Acetyltransferases 1 and Acetyl-CoA Acetyltransferases 2

ACAT has two isoforms: ACAT1 (which is expressed in macrophages, VSMCs, and other cell types) and ACAT2 (mainly expressed in the intestinal enterocytes, macrophages, and hepatocytes). ACAT2 has 44% sequence similarity with ACAT1 (Cases et al., 1998; Lee et al., 2000b; Sakashita et al., 2003; Allahverdian et al., 2012). ACAT1 was first identified in 1993 by Chang et al. (1993) and is the well-studied ACAT isoform in mediating CE synthesis. ACAT1 is located in chromosome 11 (11q22.3). Human ACAT1 gene spans 27 kb and has 12 exons and 11 introns. ACAT1 gene encodes a 45.1-kDa product, which is composed of 427 amino acids. ACAT1 was predominantly expressed in plaque macrophages in human patients, and its expression was significantly increased during monocyte differentiation to macrophages (Miyazaki et al., 1998). Moreover, several proatherogenic stimuli, including interferon-γ (Panousis and Zuckerman, 2000b), oxLDL (Wang et al., 2016b), TNF-α (Lei et al., 2009), insulin (O’Rourke et al., 2002), leptin (O’Rourke et al., 2002), Chlamydia pneumoniae infection (Liu et al., 2010a), and asymmetric dimethylarginine (Zhu et al., 2010), upregulated the expression/activity of ACAT1 in macrophages. Furthermore, cholesterol loading of aortic VSMCs also leads to increase in ACAT1 activity, while ACAT2 was not expressed by arterial VSMCs (Rong et al., 2005). Also, oxLDL increases the ACAT1-dependent foam cell formation in VSMCs via toll-like receptor 4 (TLR4)/MyD88/NF-κB-mediated proinflammatory pathways (Yin et al., 2014). In contrast, the ACAT1 expression in macrophages can be decreased by ghrelin [via growth hormone secretagogue receptor (Wan et al., 2009)], incretins [via cyclic AMP (cAMP) activation (Nagashima et al., 2011)], and the vasoprotective gasotransmitter hydrogen sulfide (H₂S) [via K_ATP/ERK1/2 pathway (Zhao et al., 2011)]. The expression of ACAT1 can be regulated by a few miRNAs, including miRNA-9 (Xu et al., 2013a), miRNA-27a/b (Zhang et al., 2014), and miRNA-467b (Wang et al., 2017a), all of which reduced the macrophage-derived foam cell formation. Clinically relevant, two genetic variants of ACAT1 gene (such as rs1154556 and rs10913733) were associated with an increased risk of CAD in Chinese population (Wang et al., 2017i).

A large part of the scientific knowledge about ACAT1 and ACAT2 has been gained from genetically engineered mice by breeding with atherosusceptible mouse strains. Total deficiency or liver-specific deletion (by administering antisense oligonucleotide targeting ACAT2) of ACAT2 has consistently reduced atherosclerosis in mice (Willner et al., 2003; Bell et al., 2006, 2007), whereas the outcome of deletion/pharmacological inhibition of ACAT1 in experimental atherosclerosis is controversial (Rudel et al., 2005; Farese, 2006). On the one hand, global deletion of ACAT1 in either ApoE^−/− or LDLR^−/− mice show reduced CE accumulation and atherosclerosis development, but caused cutaneous xanthomatosis and dry eye in mice, probably due to an excessive FC accumulation (Yagyu et al., 2000). Similarly, myeloid-specific ACAT1 deficiency (by breeding with LyzM-Cre mice) reduces the content of CE and leukocyte adhesion to activated endothelium, lesional macrophage content, and atherosclerosis plaque area in ApoE^−/− mice, without causing common side effects caused by the ACAT1 total deficiency (Huang et al., 2016b). Myeloid-specific ACAT1 ablation also reduces macrophage inflammation and prevents diet-induced obesity (Huang et al., 2018). On the other hand, myeloid cell-specific deletion of ACAT1 (by bone marrow transplantation) unexpectedly show accelerated atherosclerosis in both ApoE^−/− (Su et al., 2005) and LDLR^−/− mice (Fazio et al., 2001). Pharmacological inhibition of ACAT by inhibitors increases atherosclerotic lesion area in ApoE^−/− mouse and rabbit models of atherogenesis (Perrey et al., 2001). These unexpected findings are probably due to a FC-induced cytotoxicity and an impairment of cholesterol efflux (Fazio and Linton, 2006).

Diverse pharmacological inhibitors of ACAT have been explored as effective therapeutic strategies to assess the therapeutic potential of ACAT inhibition in atherosclerosis. Most of these inhibitors have shown promising results in ameliorating atherosclerosis development in animal models. For example, ACAT inhibitors, such as avasimibe (Nicolosi et al., 1998; Delsing et al., 2001; Kharbanda et al., 2005), CS-505 (Terasaka et al., 2007), T-2591 (Yasuhara et al., 1997), HL-004 (Ishii et al., 1998), K604 (Yoshinaka et al., 2010), F1394 (Rong et al., 2013), and tomatidine (Fujiwara et al., 2012), have been reported to inhibit foam cell formation and atherosclerosis in hyperlipidemic mice. Some ACAT inhibitors, like avasimibe, have been shown to reduce the systemic inflammation and improve the endothelial functions in patients with hypercholesterolemia (Kharbanda et al., 2005). Most of these inhibitors partially inhibited the ACAT activity and both isoforms of ACAT. Recently, Shibuya et al. (2018) identified a highly potent ACAT1 inhibitor (IC₅₀ = 4.0 nM), which reduced lipid-accumulation in the aortic arch of hamsters fed with an atherogenic diet, raising the therapeutic potential of this compound to attenuate atherosclerosis in other experimental animal models and human patients with atherosclerosis. Other recent studies have shown that other treatment options, such as losartan (Rafatian et al., 2013) or inhibition of the NLRP3 inflammasome (by compound MCC950) (Chen et al., 2018b), inhibit the ACAT activity, CE accumulation, and foam cell formation. However, three randomized, double-blind, placebo-controlled trials failed to observe significant differences in carotid as well as coronary atherosclerosis between treatment with ACAT inhibitors (by pactimibe and avasimibe) and placebo (Tardif et al., 2004; Nissen et al., 2006; Meuwese et al., 2009). The evidence indicates that selective ACAT inhibition might be desired and should be used in combination with therapies that increase cholesterol acceptors (such as HDL or ApoA-1 targeted therapies), driving out excessive FC of the plaques (Fazio and Linton, 2006).

2. Neutral Cholesterol Ester Hydrolase 1

NCEH1 gene is located at chromosome 3 (3q26.31) in the ER. Compared with HSL, NCEH1 contributes more to CE hydrolysis in macrophages (Sakai et al., 2014). NCEH1 gene yields four isoforms that result from differences in mRNA cleaving. Structurally, NCEH1 is a membrane protein consisting of an N-terminal transmembrane domain, a catalytic domain, and C-terminal lipid-binding domains (Okazaki et al., 2008). The N-terminal domain has an ER-anchoring sequence. NCEH1 gene was first cloned and biologically characterized in macrophages (Ghosh, 2000; Okazaki et al., 2008). It was recently shown that estrogen significantly increases the activity of NCEH1 in mouse macrophages, suggesting the possibility of resulting sex difference of atherosclerosis development (Chiba et al., 2011).

To dissect the role of NCEH1 in foam cell formation and atherosclerosis, Igarashi et al. (2010) showed that NCEH overexpression enhanced the CE hydrolysis and promoted cholesterol efflux from macrophages. In contrast, the NCEH1 depletion promoted foam cell formation, indicating the critical role of NCEH1 in maintaining cholesterol homeostasis (Okazaki et al., 2008). In vivo, NCEH1 deletion accelerated foam cell formation and increased atherosclerotic plaque area in ApoE^−/− mice without impacting lipid profile. NCEH1 and HSL1 have comparable NCEH activity in macrophages despite one contradictory report (Buchebner et al., 2010), which showed that HSL deficiency, but not NCEH1 deficiency, abolished the NCEH activity. Mice with deficiency of both NCEH1 and HSL1 showed exaggerated atherosclerotic plaque increase in an additive manner (Sekiya et al., 2009). In contrast, mice overproducing both NCEH and ApoA4 (as cholesterol acceptor) showed decreased atherosclerosis development (Choy et al., 2003). Also, macrophage-specific overexpression of NCEH1 reduced atherosclerotic lesion area and plaque necrosis in LDLR^−/− mice due to enhanced FC efflux and reverse cholesterol transport (RCT) (Zhao et al., 2007). Excessive accumulation of FC or oxysterols induces macrophage apoptosis and atherosclerosis. NCEH1-deficient, but not HSL-deficient, macrophages were more sensitive to 25-hydroxycholesterol-induced apoptosis (Sekiya et al., 2014). Therefore, NCEH1 not only reduced the CE content in foam cells, but also prevented the oxysterols-induced apoptosis in atherosclerosis (Sekiya et al., 2014). Overall, these results highlight NCEH1 as a promising target for treating atherosclerosis.

3. Lysosomal Acid Lipase (Lipase A) and Hormone-sensitive Lipase 1 (Lipase E)

In addition to NCEH1, LAL is also a principal lipase that hydrolyzes lysosomal CE derived from LDL and modified LDL (Dubland and Francis, 2015). Genome-wide association studies identified several genetic variants of LAL gene, which was associated with increased risk for CAD (Morris et al., 2017). In mice, intravenous administration of recombinant LAL reduced coronary and aortic atheromatous lesions in LDLR^−/− mice fed an atherogenic diet (Du et al., 2004). A recent study has shown that LAL promotes RCT in cultured macrophages and in mice by increasing the expression of ABCA1 and ABCG1 (Bowden et al., 2018).

Unlike LAL, HSL is a neutral lipase expressed in macrophages. Macrophage-specific HSL-transgenic mice show enlarged aortic lesions and increased lipid accumulation in coronary arteries (Escary et al., 1999). However, the overexpression of cholesterol acceptors (ApoA4) reduces atherosclerosis in HSL-transgenic mice (Choy et al., 2003). Studies using macrophages and LDLR^−/− mice have indicated that HSL has comparable neutral CE hydrolase activity to those of NCEH1. The deletion of bone marrow-derived HSL increased a diet-induced atherosclerosis (Sekiya et al., 2009).

To date, pharmacological activators of LAL and HSL1 are not available. The specific role of LAL and HSL in foam cell formation of macrophages and VSMCs, as well as eventual effect on atherosclerosis remains to be investigated in future studies.

1. Aqueous Diffusion Efflux Pathway

Aqueous diffusion efflux pathway recently was reviewed elsewhere (Phillips, 2014). This pathway involves a simple diffusion process, representing in this way the nonprotein-mediated cholesterol efflux pathway. It was shown that it is one major contributor to cholesterol efflux particularly in normal mouse peritoneal macrophages (MPMs) (∼80%) (Adorni et al., 2007). However, in cholesterol-loaded MPMs, aqueous diffusion was not changed and its contribution to cholesterol efflux became smaller (Adorni et al., 2007). In addition, the major variations in cellular cholesterol efflux rates are not due to aqueous diffusion efflux pathway, but to membrane transporters-mediated cholesterol efflux pathways (Phillips, 2014).

2. Transporter-Dependent Cholesterol Efflux Pathway

A significant pathway for cholesterol efflux from macrophages involves the interaction between transporters (ABCA1 and ABCG1), SR-BI, and acceptors (ApoA-1, HDL, and ApoE) (Brunham et al., 2006; Rosenson et al., 2011). It is proposed that ABCA1 and ABCG1 can act in a sequential way, in which ABCA1 generates nascent HDL, which then further promotes cholesterol efflux via ABCG1 (Gelissen et al., 2006). Genetic knockdown studies indicate that ABCG1 and ABCA1 account for about 20% and 50% of the net cholesterol efflux from cholesterol-enriched MPMs, respectively (Adorni et al., 2007). Furthermore, both ABCA1 and ABCG1 together account for about 60%–70% of the net cholesterol efflux to serum or HDL from LXR-activated macrophages loaded with cholesterol (Yvan-Charvet et al., 2007a). This part of the review will focus on current understanding of the function and regulation of these transporters (ABCA1 and ABCG1), SR-BI, and the acceptors (ApoA-1, HDL, and ApoE), which are related to cholesterol efflux from macrophages.

a. ATP-binding Cassette Transporter A1

ABCA1 is a member of the ABC transporter superfamily that utilizes ATP as a source of energy to transport various substrates across cellular membranes (Dean et al., 2001). ABCA1, originally named as ABC1, was identified by a PCR-based approach and cloned in 1994 (Luciani et al., 1994). The ABCA1 gene consists of 50 exons spanning 149 kb and encodes a 2261 amino acid integral membrane protein, consisting of two transmembrane domains (TMDs) with six helices each and two nucleotide-binding domains (NBDs) (Santamarina-Fojo et al., 2000; Wang and Smith, 2014). The NBDs exhibit a nucleotide-free state, while the two TMDs link each other via a narrow interface in the membrane. Additionally, two extracellular domains of ABCA1 form an elongated hydrophobic tunnel. There are two large extracellular loops that are linked by a disulfide linkage (Fitzgerald et al., 2002; Qian et al., 2017).

It was not until 1999 that mutations of ABCA1 gene were identified as underlying the etiology of Tangier disease (Rust et al., 1999), which is characterized in the homozygous state by an HDL deficiency, frequently premature CAD, extremely enlarged yellow tonsils, and clouding of the cornea (Bale et al., 1971; Serfaty-Lacrosniere et al., 1994; Rust et al., 1999). Further studies indicate that ABCA1 plays a major role in HDL biosynthesis by mediating FC and phospholipid efflux to lipid-poor ApoA-1, thereby producing nascent HDL particles in the liver (Yokoyama, 2006; Phillips, 2014). Fibroblasts from subjects with Tangier disease are deficient in cholesterol efflux to ApoA-1 (Remaley et al., 1997), indicating that ABCA1 is an important transporter mediating the cholesterol efflux. Details about the interaction of the ABCA1 protein with ApoA-1 in mediation of the cholesterol efflux were reviewed recently (Wang and Smith, 2014; Phillips, 2018). To date, there is no clear consensus regarding the process by which the ABCA1 protein regulates the transport of cholesterol and lipoprotein from the plasma membrane to the acceptor ApoA-1.

Consistent with the proposed function of ABCA1 in nascent HDL formation, ABCA1-deficient mice have almost undetectable levels of HDL, with a significant decrease in total plasma cholesterol and phospholipids (McNeish et al., 2000; Orsó et al., 2000). Overexpression of ABCA1 in mice led to increased HDL-cholesterol (HDL-C) and ApoA-1 levels, facilitating hepatic RCT and biliary cholesterol excretion (Singaraja et al., 2001; Vaisman et al., 2001). However, the studies in vivo from some groups have led to opposite results about the role of ABCA1 expression in atherosclerosis development. Complete ABCA1 deficiency in wild-type, LDLR^−/−, and ApoE^−/− mice did not alter progression of atherosclerosis (McNeish et al., 2000), but repopulation of ABCA1^−/− mice with wild-type macrophages (McNeish et al., 2000) or macrophages from ABCA1-overexpressing mice (McNeish et al., 2000) led to a significant decrease of atherosclerotic lesion in mice. In addition to lipid regulation, ABCA1 protein is also involved in the regulation of apoptosis and inflammation (Soumian et al., 2005). ABCA1 executes its anti-inflammatory effects by modifying cell membrane lipid rafts and directly activating signaling pathways, including Janus kinase 2/STAT3, protein kinase A (PKA), Rho family G protein CDC42, and PKC (Liu and Tang, 2012).

ABCA1 protein plays a critically important role in cholesterol homeostasis and may be viewed as a protector against atherosclerosis, thus there are many studies to decipher how its expression is regulated at both transcriptional and posttranscriptional levels (Schmitz and Langmann, 2005; Zarubica et al., 2007) (Fig. 3). ABCA1 expression is regulated in a tight pathway with a short protein half-life (1 to 2 hours) (Yokoyama et al., 2012) and rapid turnover in macrophages (Soumian et al., 2005). ABCA1 protein expression is highly regulated by a variety of molecules, including secondary messengers (e.g., cAMP), nuclear receptors [e.g., LXR, RXR, PPAR, pregnane X receptor (PXR)], and cytokines [e.g., TNF-α, transforming growth factor-β (TGF-β), interleukin-1β (IL-1β)] (Zarubica et al., 2007). cAMP upregulates the expression of ABCA1 protein by acting at both the transcriptional and translational levels (Haidar et al., 2002; Denis et al., 2003). Nuclear receptors are ligand-dependent transcriptional factors that mediate the expression of their target genes (Beaven and Tontonoz, 2006). The activation of LXR and RXR by physiologically occurring oxysterols (e.g., 27-hydroxycholesterol), retinoids, or synthetic agonists stimulates the transcription of ABCA1 via the DR4 element in the ABCA1 promoter (Zarubica et al., 2007). PPAR upregulates ABCA1 expression and RCT indirectly via enhancement of the transcription of LXRα (Chinetti et al., 2001). PXRs, regulated by many compounds including both natural and synthetic steroids, downregulate the ABCA1 gene transcription (Sporstol et al., 2005). Cytokines were shown to exhibit pleiotropic and contradictory effects on ABCA1 expression by cross-talks between the cellular process of cholesterol and inflammatory reactions (Zarubica et al., 2007). TNF-α, IL-1β, and interferon-γ downregulate the LXR-mediated increase of ABCA1 protein expression (Lusis, 2000; Panousis and Zuckerman, 2000a), whereas TGF-β positively regulates the ABCA1 expression (Panousis et al., 2001). Posttranscriptional regulation also plays a critical role in the regulation of ABCA1 protein levels by adjusting either protein stability or its transporter activity (Llaverias et al., 2005). It was reported that the protein stability of ABCA1 is regulated by the proteasomal, lysosomal, and calpain systems (Ogura et al., 2011; Yokoyama et al., 2012; Aleidi et al., 2015), whereas its activity is under the fine control of diverse protein kinases, such as PKA and protein kinase CK2 (Haidar et al., 2004; Roosbeek et al., 2004). It was shown that a proline, glutamic acid, serine, and threonine sequence in ABCA1 is very important for its degradation by calpain protease, since deletion of this motif leads to increase ABCA1 protein levels by four- to fivefold (Wang et al., 2003).

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Fig. 3.

Regulation of ABCA1 expression at both transcriptional and post-transcriptional levels. At the transcriptional level, ABCA1 protein expression is highly regulated by a variety of molecules, including secondary messengers [e.g., cyclic AMP (cAMP)], nuclear receptors (e.g., LXR, RXR, PPAR, and PXR), and cytokines [e.g., TNF-α, transforming growth factor-β (TGF-β), interleukin-1β (IL-1β)]. At the post-transcriptional level, the protein stability of ABCA1 is governed by the proteasomal, lysosomal, and calpain systems. Also, miRNA-10b, miRNA-23a-5p, miRNA-33, miRNA-106b, miRNA-128-2, miRNA-148a, miRNA-183, and miRNA-758 have been reported to regulate ABCA1 protein expression. The long noncoding RNAs (lncRNAs), including lncRNA MeXis, long intervening lincRNA-DYNLRB2-2, lnc-HC, and lncRNA RP5-833A20.1, have also been involved in the regulation of ABCA1 expression (green arrows: upregulation; red lines: downregulation).

The regulation of ABCA1 protein expression by miRNA was recently investigated. miRNAs, are a class of ncRNAs that are predominantly posttranscriptional regulators of gene expression (Ambros, 2004; Bartel, 2009). To date, miRNA-10b (Wang et al., 2012a), miRNA-23a-5p, miRNA-33 (Rayner et al., 2010), miRNA-106b (Kim et al., 2012), miRNA-128-2 (Adlakha et al., 2013), miRNA-148a (Goedeke et al., 2015), miRNA-183 (Sarver et al., 2010), and miRNA-758 (Ramirez et al., 2011) have been reported to regulate the ABCA1 protein expression. MiRNA-10b directly repressed ABCA1 expression and also suppressed cholesterol efflux from both murine and human macrophages (Wang et al., 2012a). Transfection of miRNA-23a-5p inhibitor enhanced cholesterol efflux and decreased foam cell formation through upregulating the ABCA1 expression levels. miRNA-23a-5p reduced ABCA1 expression via repressing the 3′-untranslated regions (UTR) activity of the ABCA1 transcripts. Long-term in vivo systemically delivered miRNA-23a-5p antagomir significantly increased ABCA1 expression, reduced atherosclerosis development, and promoted plaque stability in the aorta of ApoE^−/− mice (Yang et al., 2018a). miRNA-33, located within the gene encoding sterol regulatory element-binding factor-2, inhibited ABCA1 expression, thus resulting in the decreased cholesterol efflux (Rayner et al., 2010). miRNA-128-2 inhibited the ABCA1 expression directly via binding to its 3′-UTR. The administration of miRNA-128-2 led to the decrease both protein and mRNA levels of ABCA1 (Adlakha et al., 2013). miRNA-148a decreased expression of ABCA1 and circulating HDL-C levels in vivo (Goedeke et al., 2015). miRNA-758 directly targeted the 3′-UTR of ABCA1, repressed the expression of ABCA1, and reduced cellular cholesterol efflux to ApoA-1. Conversely, the application of anti-miRNA-758 increased ABCA1 expression (Ramirez et al., 2011). miRNA-106b significantly decreased the ABCA1 levels and the cholesterol efflux in neuronal cells (Kim et al., 2012). miRNA-183 targeted the 3′-UTR of ABCA1 mRNA to degrade ABCA1 mRNA in the colon cancer cells and other tumor cells (Sarver et al., 2010). There has been no further study to investigate the effect of miRNA-106b and miRNA-183 on macrophages and liver cells so far.

Recent studies also showed that lncRNA, a large subgroup of RNAs that are >200 nucleotides. Although most lncRNAs do not have apparent protein coding potential, lncRNAs also play a very important role in modulating ABCA1 protein expression. lncRNAs can regulate the ABCA1 expression at both transcriptional and posttranscriptional levels. It was reported that lncRNA macrophage-expressed LXR-induced sequence (MeXis) increased the ABCA1 protein expression at the transcriptional level by interacting with and guiding promoter to bind to the transcriptional coactivator DDX17 in macrophages (Sallam et al., 2018). Furthermore, the loss of MeXis in mouse bone marrow cells impaired the cellular reaction to cholesterol overload and accelerated the atherosclerotic development (Sallam et al., 2018). Increased long intervening noncoding RNA-DYNLRB2-2 expression promoted the ABCA1-mediated cholesterol efflux by upregulation of the ABCA1 expression through the glucagon-like peptide-1 (GLP-1) receptor signaling pathway (Hu et al., 2014) and decrease of TLR2 expression (Li et al., 2018c) in macrophages. A newly identified lncRNA named lnc-HC, which interacts with hnRNPA2B1 to form an lnc-HC-hnRNPA2B1 complex, decreased the ABCA1 expression at the posttranscriptional level within hepatocytes (Lan et al., 2016). However, the effect of lnc-HC on ABCA1 expression in macrophages remains to be examined. On the contrary, another lncRNA, RP5-833A20.1, attenuated the ABCA1 levels, thus reducing the cholesterol efflux via the miRNA-382-mediated NFIA pathway (Hu et al., 2015).

b. ATP-binding Cassette Transporter G1

ABCG1 is another member of the ABC superfamily of transporters (Tarr et al., 2009; Tarling and Edwards, 2012). ABCG1 is a half transporter that contains a single ABC and a single TMD/6 transmembrane α-helice on one polypeptide chain (Tarling, 2013). The half transporter ABCG1 is generally considered to function by forming homodimers (ABCG1:ABCG1) to transport substrates across the cell membrane (Tarling and Edwards, 2011). ABCG1 was first described and cloned in 1996 as the human homolog of the Drosophila white gene (Chen et al., 1996). After that, it took around 4 years until ABCG1 again received intensive attention because it is similar to ABCA1 in the expression pattern in monocytes (Schmitz et al., 2001). ABCG1 is involved in regulation of intracellular cholesterol homeostasis and cholesterol efflux from cells to HDL particles for RCT (Rosenson et al., 2012). ABCG1 also effluxes cholesterol to LDL, liposomes, and cyclodextrin (Wang et al., 2004; Kennedy et al., 2005) and it exports sphingomyelin, phosphatidylcholine, and oxysterols to HDL and albumin (Kobayashi et al., 2006; Xu et al., 2009). In addition to regulation of cholesterol efflux from cells, ABCG1 protein exhibits other physiologic functions as well (Sano et al., 2014). For example, ABCG1 is involved in cell proliferation, apoptosis, and immune response (Sano et al., 2014). It has been shown that ABCG1 suppresses the proliferation of T cells by LXR signaling (Bensinger et al., 2008). ABCG1 also induced the apoptosis of cultured cells (Seres et al., 2008), but inhibited the apoptosis of macrophages and prostate cancer cells by decreasing signaling of TLR4 and NADPH oxidase 2 (Yvan-Charvet et al., 2010a) and downregulating Akt signaling (Yvan-Charvet et al., 2010a), respectively.

Consistent with the function of ABCG1 in regulation of macrophage cholesterol efflux from cultured macrophages, ABCG1-deficient mice, first described in 2005, when fed a Western-type diet, displayed excessive lipid accumulation in macrophages within multiple organs, particularly in the lung (Kennedy et al., 2005). Unexpectedly, whole body loss of ABCG1 or overexpression of human ABCG1 had no influence on plasma lipid or lipoprotein levels (Kennedy et al., 2005; Burgess et al., 2008), which are in stark contrast to loss of ABCA1. Deeper insights about the coordinated participation of ABCA1 and ABCG1 in regulation of cholesterol efflux from macrophage have been obtained from animal studies (Rosenson et al., 2012). The combined deficiency of ABCA1 and ABCG1 resulted in markedly accelerated atherosclerotic lesion development in mice compared with the deficiency of either ABCA1 or ABCG1 (Yvan-Charvet et al., 2007b; Out et al., 2008). ABCA1 and ABCG1 double knockout macrophages showed apparently defective cholesterol efflux to HDL and ApoA-1 compared with either ABCA1 or ABCG1 knockout macrophages (Yvan-Charvet et al., 2007b; Out et al., 2008).

ABCG1 protein expression is mediated at both the transcriptional and posttranscriptional levels (Fig. 4). At the transcriptional level, ABCG1 shares the common regulatory pathways of gene expression with ABCA1. The ABCG1 expression is upregulated by the nuclear receptors LXR, RXR, and PPAR activated by their agonists (oxysterols, retinoids, fatty acids, or synthetic agonists) (Venkateswaran et al., 2000; Hardy et al., 2017). Upregulation of ABCG1 expression mediated by LXR agonists probably involves the presence of multiple LXR responsive elements by the ABCG1 gene promoter and likely only requires the isoform LXRα in human macrophages (Sabol et al., 2005; Ishibashi et al., 2013). Further study suggests that LXR recruitment at the human ABCG1 locus is promoted by the G protein pathway suppressor 2 (Jakobsson et al., 2009). ABCG1 in macrophages is also transcriptionally regulated by the PPARγ-LXR pathway (Li et al., 2004a). Retinoic acid receptor/RXR heterodimer can bind LXR responsive elements in ABCG1 promoters and transactivates ABCG1 in macrophages as well (Ayaori et al., 2012).

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Fig. 4.

Regulation of ABCG1 expression at both transcriptional and post-transcriptional levels. At the transcriptional level, ABCG1 shares common gene expression regulatory pathways with ABCA1. The ABCG1 expression is upregulated by the nuclear receptors LXR, RXR, and PPAR activated by their agonists (oxysterols, retinoids, fatty acids, or diverse synthetic agonists). At the post-transcriptional level, the protein stability of ABCA1 is governed by the proteasomal and calpain systems. Also, miRNA-10b, miRNA-23a-5p, miRNA-33, miRNA-128-2, miRNA-146a-5p, and miRNA-378 are involved in the regulation of ABCG1 protein (green arrows: upregulation; red lines: downregulation).

Other mechanisms also contribute to the stability and activity of human ABCG1. Compared with ABCA1, there is limited evidence concerning the posttranscriptional regulation of ABCG1 or protein-protein interactions involving ABCG1. It was reported that calpain facilitated ABCG1 degradation by slicing ABCG1 on the cell surface (Adlakha et al., 2013). The proteasomal inhibition prevented degradation of ABCG1 and led to the accumulation of phosphorylated ABCG1 (Nagelin et al., 2009). Degradation of ABCG1 is suggested to be mediated via the ubiquitin-proteasome system-mediated non-lysosomal pathways (Nakaya et al., 2017) by the NEDD4-1 (neural precursor cell-expressed developmentally downregulated gene 4) and E3 ubiquitin ligases HUWE1 (HECT, UBA, and WWE domain containing 1 E3 ubiquitin protein ligase) (Aleidi et al., 2015). ABCG1 ubiquitination and its proteasomal degradation can be inhibited by cholesterol through interplay with a cholesterol recognition/interaction amino acid consensus (CRAC) motif located in the ABCG1 transmembrane domain (Hsieh et al., 2014; Sharpe et al., 2015).

ABCG1 protein expression was also recently investigated in relation to regulation by miRNAs. The miRNAs implicated in ABCG1 protein regulation include miRNA-10b (Wang et al., 2012a), miRNA-23a-5p, miRNA-33, miRNA-128-2, miRNA-146a-5p, and miRNA-378. The inhibition of miRNA-23a-5p enhanced cholesterol efflux and decreased foam cell formation through upregulating ABCG1 expression levels. miRNA-10b repressed ABCA1 expression and downregulated cholesterol efflux from murine or human macrophages (Wang et al., 2012a). Long-term in vivo systemically delivered miRNA-23a-5p antagomir significantly increased the ABCG1 expression in the aorta of ApoE^−/− mice (Yang et al., 2018a). miRNA-33 inhibited ABCG1 expression, thus resulting in decreasing cholesterol efflux (Rayner et al., 2010). Upregulation of miRNA-33a-5p stimulated by inflammatory cytokines (i.e., IL-6, TNF-α) inhibited the ABCG1-mediated cholesterol efflux from THP-1 macrophages (Mao et al., 2014). miRNA-128-2 inhibited the expression of ABCG1 directly. The administration of miRNA-128-2 led to decrease in the mRNA and protein levels of ABCG1 in mice (Adlakha et al., 2013). Elevated miRNA-146a-5p antagonized the increase of ABCG1 in low-dose LPS-tolerized cells (Li et al., 2015b). The decrease of miRNA-378 level enhanced ABCG1-mediated macrophage cholesterol efflux to HDL by inducing ABCG1 protein expression (Wang et al., 2014a).

c. Scavenger Receptor Class B Type 1

SR-BI is an 82-kDa integral membrane protein, which (together with lysosomal integral membrane protein-2) is a member of the CD36 superfamily of scavenger receptor proteins (Phillips, 2014). SR-BI has a hairpin-looped structure with two short N- and C-terminal transmembrane domains, two cytoplasmic tails, and a large extracellular domain (Williams et al., 1999; Meyer et al., 2013). In 1996, it was identified that SR-BI is an HDL receptor that regulates cholesterol uptake into liver cells (Acton et al., 1996). This process selectively transports the CE from mature HDL into cells without endocytosis and degradation of the HDL particles (Acton et al., 1996). This receptor is expressed primarily in liver, where it acts in the RCT pathway (Zannis et al., 2006). In addition to promotion of delivery of HDL-C to cells, SR-BI increases the efflux of cellular cholesterol to HDL (Ji et al., 1997; Jian et al., 1998). When incubated with synthetic cholesterol-free HDL, SR-BI-transfected Chinese hamster ovary cells increased initial rates of efflux by approximately threefold compared with control cells, suggesting that SR-BI expression enhanced net cholesterol efflux regulated by HDL (Ji et al., 1997). However, compared with ABCA1 and ABCG1, the contribution of SR-BI to efflux from macrophages is small (Adorni et al., 2007). The performed studies indicated that the SR-BI is a multifunctional receptor that regulates bidirectional flux of lipids, which might be dependent on the content of cholesterol in cells (Rosenson et al., 2012).

In addition to regulating lipid metabolism, SR-BI can also mediate inflammatory responses. SR-BI interaction with HDL reduced the inflammatory response to LPS in human macrophages by markedly reducing NF-κB activation (Song et al., 2015). Furthermore, recent studies have shown that the interaction of macrophage SR-BI with apoptotic cells activated phosphoinositide 3-kinase (PI3K)/Akt signaling and induced the expression of anti-inflammatory cytokines (Tao et al., 2015a). HDL activated PI3K/Akt signaling in macrophages, which is mediated by SR-BI and involves interaction with its adaptor protein PDZK1 (PDZ domain-containing 1) and activation of sphingosine 1-phosphate receptor 1 signaling (Al-Jarallah et al., 2014). SR-BI interaction with HDL also prevents endothelial cell inflammation reaction by regulating eNOS activation and expression of the antioxidant enzyme 3-beta-hydroxysteroid-delta 24-reductase (Yuhanna et al., 2001; McGrath et al., 2009). SR-BI-mediated production of NO and 3-beta-hydroxysteroid-delta 24-reductase lead to alleviation of TNF-α-stimulated NF-κB activation, resulting in the reduced expression of inflammatory monocyte adherence proteins and chemokines in endothelial cells, thereby reducing monocyte recruitment into the intima (Bess et al., 2011). Additionally, a major portion of the endothelial cell-mediated transcytosis of HDL from the apical to the basolateral side is mediated by SR-BI, suggesting that SR-BI regulated HDL transport to the subendothelium, increasing cholesterol efflux from macrophages (Rohrer et al., 2009; Vaisman et al., 2015).

In vivo studies showed that SR-BI deficiency in the bone marrow led to an accelerated atherosclerosis despite increased plasma HDL-C level, which indicated that it played a critical role in the HDL metabolism and exhibited atheroprotective effects in mice (Covey et al., 2003; Rigotti et al., 2003; Van Eck et al., 2004; Brundert et al., 2006). SR-BI overexpression in bone marrow-derived cells protected against atherosclerosis in LDLR^−/− mice, as well as in ApoE^−/− mice (Covey et al., 2003; Zhang et al., 2003a). However, the physiologic effect of SR-BI-mediated arterial macrophage-specific cholesterol efflux in vivo is not clear (Brundert et al., 2006; Burgess et al., 2008; Yvan-Charvet et al., 2008). Some studies showed that a selective uptake of HDL-CE and the cholesterol efflux from MPMs are independent of SR-BI (Brundert et al., 2006). SR-BI in primary macrophages, although increasing cholesterol efflux in vitro, did not contribute to macrophage RCT in vivo (Wang et al., 2007a). Hepatic SR-BI modulates the changes in the composition and structure of HDL (El Bouhassani et al., 2011). It was indicated that SR-BI polymorphisms contributed to the functional potential of the cholesterol disposition pathway (Vergeer et al., 2011), suggesting that this receptor is involved in RCT and atherosclerosis. Human studies show that carriers of the mutation of SR-BI in humans (P297S mutation) had higher HDL-C levels, but decreased the potential for cholesterol efflux from macrophages without aggravation of atherosclerosis (Alam et al., 2001). In addition, a recent study has shown that endothelial SR-BI promoted LDL transcytosis via dedicator of cytokinesis (DOCK4) and ensuing circulating LDL entry into and retention in the artery wall to instigate atherosclerosis (Huang et al., 2019). The differential functions of SR-BI in macrophage and endothelial cells suggest the necessity to target SR-BI in a specific cell type to achieve atheroprotection.

SR-BI expression is mediated by both transcriptional and posttranscriptional mechanisms (Fig. 5). Many molecules have been proposed to be involved in the regulation of SR-BI expression at the transcriptional level, including nuclear transcription factors [i.e., PPAR (Straus and Glass, 2007), LXR (Malerød et al., 2002), RXR, farnesoid X receptor (Malerød et al., 2005), PXR (Sporstol et al., 2005), estrogen receptors (Stangl et al., 2002), sterol regulatory element-binding proteins (SREBPs) (Lopez and McLean, 1999)] and some endogenous factors [e.g., IGF-1 (Cao et al., 2004), p38-mitogen-activated protein kinase (MAPK) cascade (Murao et al., 2008; Leiva et al., 2011]. Recent progress toward understanding the mechanisms of regulating the SR-BI expression at the transcriptional level was summarized in several reviews (Leiva et al., 2011; Shen et al., 2018a,b).

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Fig. 5.

Regulation of SR-BI expression at both transcriptional and post-transcriptional levels. At the transcriptional level, SR-BI protein expression is regulated by a variety of molecules, including nuclear transcription factors [such as the heterodimeric nuclear receptors PPAR, LXR, RXR, FXR (farnesoid X receptor), and PXR], estrogen receptors, sterol regulatory element-binding proteins (SREBPs), and other endogenous signaling factors (e.g., IGF-1 and p38-MAPK cascade). At the post-transcriptional level, SR-BI can be regulated by estrogens insulin and glucagon as well as other hormones [e.g., triiodothyronin (T3) and thyromimetics]. In addition, miRNA-96, miRNA-125a, miRNA-185, miRNA-455, and miRNA-223 are involved in SR-BI protein regulation. (green arrows: upregulation; red lines: downregulation).

SR-BI can also be regulated posttranscriptionally, for example, by hormones such as estrogens (Zhang et al., 2007), triiodothyronine and thyromimetics (Johansson et al., 2005; Tancevski et al., 2010), insulin (Shetty et al., 2006), as well as glucagon (Nakamura et al., 2005). One important component of the posttranscriptional regulation of SR-BI is the scaffolding protein PDZK1, which regulates the SR-BI protein stability (Nakamura et al., 2005). The exact mechanisms by which the PDZK1 regulates SR-BI membrane expression remain unknown (Leiva et al., 2011). PDZK1/Na⁺/H⁺ exchanger regulatory factor 3 (NHERF3), NHERF1, and NHERF2 were shown to regulate hepatic SR-BI stability (Kocher et al., 2003; Hu et al., 2013b). The mechanisms involved in regulation of the SR-BI expression at the posttranscriptional level were also summarized in recently published works (Leiva et al., 2011; Shen et al., 2018a,b).

Recently, ncRNAs were also investigated in the regulation of SR-BI expression. The miRNAs, which regulate the SB-BI expression, include miRNA-96, miRNA-125a, miRNA-185, miRNA-455, and miRNA-223 (Wang et al., 2013). It was shown that miRNA-96, miRNA-185, and miRNA-223 directly bound the 3′-UTR of SR-BI mRNA to repress the level of SR-BI expression and the uptake of HDL. Furthermore, the decrease of miRNA-185 and miRNA-96 is related to the increase of SR-BI in the livers of ApoE^−/− mice with a high-fat diet (HFD) (Wang et al., 2013). Another study reported that miRNA-125a and miRNA-455 negatively regulated the SR-BI expression by binding to 3′-UTR of SR-BI mRNA (Hu et al., 2012). Recent work also demonstrated that obesity induced miRNA-24 and repressed the SR-BI expression, further influencing HDL uptake, lipid metabolism, and steroid hormone synthesis (Wang et al., 2018d). However, the effect of miRNAs on the SR-BI expression and cholesterol efflux in macrophage remains to be further studied.

3. Acceptors that Mediate Macrophage Cholesterol Efflux

1. Models for Studying Cholesterol Uptake

Lipid uptake can be assessed by neutral lipid-targeting lysochrome Oil red O staining, Nile red staining, 1,1′-dioctadecyl-3,3,3′3′-tetra-methylindocyanide percholorate (DiI)-labeled oxLDL (DiI-oxLDL), DiI-LDL, and DiI-acLDL (Xu et al., 2010), NBD-cholesterol, as well as radiolabeled LDL or derived LDL (e.g., ¹²⁵I-LDL, ¹³¹I-LDL) (Suits et al., 1989).

For Oil red O staining, macrophages are incubated with oxLDL with or without ACAT1 inhibitor in the presence or absence of tested natural products. The cells are then fixed with 4% formaldehyde and stained with Oil red O solution to identify lipid droplets containing CE to assess the amount of lipid inside the cell (lipid uptake) (Kosaka et al., 2001). Lipid accumulation in cells can be assessed by a microscope and separated into different grades according to the intracellular lipid droplet-occupied area. The percentage of foam cells is calculated by counting the total cell and foam cell numbers (Kosaka et al., 2001; Das et al., 2013). Oil red O-stained lipid droplets can also be assessed spectrophotometrically at 518 nm after lysis of the cells (Guo et al., 2006). Nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, is also an important staining method for the determination of intracellular lipid droplets by using fluorescent microscopy and flow cytofluorometry (Greenspan et al., 1985). The increase in fluorescence intensity can be quantitatively determined by the interactive laser cytometer (Koren et al., 1990). Its staining process and application are almost the same with Oil red O for staining lipids. In addition, fluorescent NBD-cholesterol [22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-Ol)] is also used to assess cholesterol uptake (Frolov et al., 2000; Sengupta et al., 2013). Cells are incubated with NBD-cholesterol and then lysed by adding methanol. Supernatant of cells lysate is used to measure fluorescence intensity by using fluorescence plate reader at emission spectra of 535 nm upon excitation at 475 nm (Frolov et al., 2000; Sengupta et al., 2013).

Staining with Oil Red O requires fixation of cells and staining in the presence of organic solvents (e.g., isopropanol) and is, therefore, limited to the analysis of dead cells (Majka et al., 2014). Water soluble Nile red can be used to stain both live and dead cells. However, its emission maximum at 528 nm may overlap with green (FITC, GFP) and yellow-orange fluorophores in flow cytometry (Majka et al., 2014). In recent years, a big progress has been made to develop new LD-specific probes [e.g., LipidTOX stains (Majka et al., 2014)], which are discussed in a recent review (Fam et al., 2018).

For cellular uptake of DiI-oxLDL, DiI-LDL, or DiI-acLDL, cells are incubated with them and then lysed (Teupser et al., 1996; Xu et al., 2010). Fluorescent intensity of the cell lysates is detected by using a microtiter plate reader with excitation-emission set at 520 and 580 nm, respectively (Teupser et al., 1996). DiI-oxLDL, DiI-LDL, or DiI-acLDL uptake in macrophages can also be evaluated by confocal microscopy and flow cytometry as described previously (Xu et al., 2010). For cellular uptake of radiolabeled LDL or derived LDL, cells are incubated with modified lipoproteins and then lysed in NaOH (Selmer et al., 1997). Internalized LDL is determined by measuring the NaOH solution in the gamma-counter (Selmer et al., 1997). In addition, foam cell formation can be indirectly evaluated by measuring the total cholesterol (TC) via using cholesterol/CE quantification kit (Das et al., 2013).

2. Models for Studying Cholesterol Efflux

One cause for foam cell formation is the inability of cells to export cholesterol to a sufficient extent (Hansson, 2005; Hansson et al., 2006), which can be assessed by quantitating the rate of cholesterol efflux from the cells. Cholesterol efflux assay is usually used to assess the influence of natural products on plasma acceptor-induced cholesterol efflux from cells (Low et al., 2012). Dr. Rothblat’s group (Rothblat et al., 1999) pioneered methodologies allowing the assessment of the capacity of human serum- or HDL-induced cholesterol efflux. In general, first of all, cells are loaded with labeled lipids to form foam cells and then incubated in serum-free medium to balance labeled cholesterol in cholesterol pools in cells (de la Llera-Moya et al., 2010; Khera et al., 2011), which can be combined with treatment or stimulation of tested natural products. Then the cells are incubated with acceptors to induce cholesterol efflux. The efflux of labeled lipids from the cells can be quantified (Low et al., 2012). In addition, some studies used ACAT inhibitor to prevent re-esterification of FC to CE (de la Llera-Moya et al., 2010; Khera et al., 2011) when loading lipids to cells or other steps (e.g., equilibration).

1. Alpinetin

Alpinetin (also known as 7-hydroxy-5-methoxyflavanone) is the main bioactive component in the seeds of Alpinia katsumadai Hayata. Alpinetin is also present in Amomum subulatum, Scutellaria rivularis, and plants from the ginger family, such as turmeric and cardamom (He et al., 2005, 2006). Recent studies have shown that alpinetin exerted multiple pharmacological properties, such as antitumor, anti-inflammatory, and antioxidative activities, and inhibition of platelet aggregation (Ramírez-Tortosa et al., 1999; Tang et al., 2012). It was shown that alpinetin (50–150 μg/ml) promoted cholesterol efflux and elevated the expression of PPARγ and LXRα in oxLDL-stimulated THP-1 macrophages and HMDMs. It also inhibited lipid accumulation by enhancing the expression of ABCA1 and ABCG1, suggesting that alpinetin may inhibit foam cell formation by targeting the PPARγ/LXRα/ABCA1/ABCG1 pathway (Jiang et al., 2015). Furthermore, it is also reported that alpinetin (50–200 μg/ml) had an anti-inflammatory effect by inhibiting the TNF-α, IL-6, and IL-1β expression in LPS-stimulated human macrophages (Hu et al., 2013a). While TNF-α reduced both ABCA1 and LXRα expression and suppressed the cholesterol efflux from monocytes (Voloshyna et al., 2014), it is possible that alpinetin may reverse the suppression of cholesterol efflux proteins ABCA1/ABCG1 and abolish the formation of foam cells by suppressing TNF-α expression.

2. Anthocyanin

Anthocyanins (ACs) are a class of water-soluble natural pigments widely present in dark-colored fruits and foods, such as purple sweet potato, grape, blueberry, black rice, and black soybean. In general, ACs comprise six important types of pigments, including geranium, cyanidin, delphinidin, peony, morning glory, and mallow pigment. Both ACs and anthocyanidins (the sugar-free counterparts of ACs), have shown various biologic activities. For example, ACs regulate blood lipids by decreasing the levels of TG, TC, and LDL-cholesterol (LDL-C), suggesting that ACs may have positive effects on CVD (Wallace et al., 2016). Anthocyanin mixture also inhibited the inflammatory response in hypercholesterolemic patients by decreasing the levels of serum high sensitivity C-reactive protein (hs-CRP) (Zhu et al., 2013).

Two bioactive ACs, cyanidin-3-glucoside (10 μM) and peonidin-3-glucoside (100 μM), significantly increased the macrophage cholesterol efflux in MPMs through the PPARγ-LXRα-ABCA1 pathway (Xia et al., 2005). Moreover, in THP-1-derived macrophages, AC-rich fraction extracted from the wild blueberry (Vaccinium angustifolium) powder reduced lipid accumulation (Del Bo’ et al., 2016). Another similar observation was recorded in human kidney 2 cells, cyanidin-3-O-β-glucoside chloride, or cyanidin chloride (50 μM) inhibited the high-glucose-induced cholesterol accumulation and inflammation by activating LXRα pathway, and activated the PPARα-LXRα-ABCA1-dependent cholesterol efflux (Du et al., 2015).

Further study indicated that the black rice AC-rich extract (300 mg/kg body weight per day) inhibited the production of oxLDL and reduced the levels of TC and LDL-C, while upregulating the HDL-C level in serum from rats and ApoE^−/− mice, and also narrowed the area of atherosclerotic plaque and improved the stability of plaque to prevent the occurrence of embolism (Xia et al., 2006). Moreover, the ACs derived from pomegranate reduced blood lipids and exhibited an anti-inflammatory activity (Wang et al., 2018b). It was reported that pomegranate peel extract containing ACs (1 g/kg diet) has a slight effect on fatty streak formation and lipid metabolism in hypercholesterolemic rabbits (Sharifiyan et al., 2016). These studies indicated that the role of an AC-rich diet on the reduction of the risk of developing CVD might possibly be due to the ability of these compounds to inhibit lipid accumulation.

3. Baicalin

Baicalin, a glucuronide of baicalein, is a flavone glycoside that is present in the root of Scutellaria baicalensis Georgi with content as high as 12.1% (Makino et al., 2008). The latter is a traditional Chinese medicine widely used to treat acute and chronic hepatitis, nephritis, and allergic diseases due to its anti-inflammatory and hypolipidemic effects. One study indicated that baicalin (50 μM) significantly increased HDL- but not ApoA1-mediated cholesterol efflux and upregulated the expression of SR-BI in a dose- and time-dependent manner (Yu et al., 2016). Another study showed that baicalin (50 and 100 μM) exerted potentially antiatherosclerotic effects by inhibition of form cell formation and lipid deposition via promoting cholesterol efflux through the PPARγ-LXRα-ABCA1/ABCG1 pathway in THP-1 macrophages (He et al., 2016b).

Baicalin also markedly decreased atherosclerotic lesion size and lipid accumulation in the carotid arteries in an atherosclerotic rabbit model (He et al., 2016b). Furthermore, long-term treatment with baicalin (80 mg/kg body weight) in rats with HFD markedly reduced the rising level of TC, LDL-C, NEFA, insulin, and TNF-α and also significantly ameliorated the fasting serum glucose levels (Guo et al., 2009), which suggests that baicalin may regulate the glucose/lipid metabolic disorders. Baicalin reduces TC, TG, LDL-C, and hs-CRP in patients with rheumatoid arthritis who have an increased risk of CAD (Hang et al., 2018).

4. Chrysin

Chrysin, also called 5,7-dihydroxyflavone, is a flavone present in blue passionflower (Passiflora caerulea), Pelargonium crispum, and Oroxylum indicum. It is one of the main active ingredients of propolis.

In a model of Wistar rats fed with HFD, chrysin (200 mg/kg body weight per day) decreased the mean levels of serum TC, TG, LDL-C, and VLDL-C (Anandhi et al., 2014). A recent report revealed that chrysin activated PPARγ, the key regulator in lipid metabolism, to exert an anti-inflammatory effect (Feng et al., 2014). This suggested that chrysin might have an antiatherosclerotic effect, possibly by decelerating the formation of foam cells. Additional study displayed that HDL-mediated RAW264.7 macrophages cholesterol efflux was significantly increased after chrysin (10 μM) treatment by the upregulation of PPARγ, LXRα, ABCA1, and ABCG1 expression (Wang et al., 2015b). Additionally, chrysin also prevented the transcription of SR-A1 and SR-A2 but had no effect on CD36, suggesting that it may inhibit the cholesterol uptake by downregulating the expression of SR-A1 and SR-A2 (Wang et al., 2015b).

5. Cyanidin-3-O-β-glucoside

As a member of blueberry AC family, cyanidin-3-O-β-glucoside (C3G) is the most widely distributed AC in nature. C3G has anti-inflammatory action, lowers the blood fat, as well as protects cardiovascular health through various mechanisms (Duchnowicz et al., 2012; Luo et al., 2012). It has been reported that C3G (0.5, 5, and 50 μM) upregulated the expression of ABCG1 and ABCA1 in a dose-dependent manner and promoted the cholesterol efflux in human aortic endothelial cells (Wang et al., 2012c), suggesting a novel mechanism by which C3G reduces oxidative damage on endothelial cells.

The antiatherosclerotic effect of C3G was recently evaluated in an HFD-induced atherosclerosis rat model. The results showed that diet supplementation with C3G (150 mg/kg) markedly decreased body weight, visceral adiposity, TG, TC, free fatty acids, and atherosclerosis index (Um et al., 2013). In ApoE^−/− mice, C3G (2 g/kg diet) prevented the hypercholesterolemia-induced endothelial dysfunction and the development of atherosclerosis by inhibiting cholesterol and 7-oxysterol accumulation in the aorta (Wang et al., 2012b). Interestingly, it was shown that protocatechuic acid, a gut microbiota metabolite of C3G in ApoE^−/− mice, displayed a promising antiatherogenic effect at physiologically reachable concentrations (0.25–1 μM), accelerating the cholesterol efflux in acLDL-loaded MPMs or THP-1 macrophages by interfering with the regulation of miRNA-10b-ABCA1/ABCG1 cascade (Wang et al., 2012a). Therefore, the gut metabolites of AC could also be explored as potential novel molecules for atherosclerotic prevention and treatment. Furthermore, a double-blind, randomized, placebo-controlled trial showed that consumption of anthocyanin increased HDL-C, decreased LDL-C, and promoted cellular cholesterol efflux in hyperlipidemia patients (Qin et al., 2009).

6. Daidzein

Daidzein is a naturally occurring isoflavone found in soybeans and other legumes. Because it is a nonsteroidal compound with estrogen-like biologic activities, daidzein is also regarded as a phytoestrogen. There are many clinical studies that investigated the effects of daidzein on the treatment of hypertension and menopausal syndrome, as well as alcoholism (Liu et al., 2014d,e). Studies conducted over the last several decades showed that it has antiosteoporosis, antioxidative, hypolipidemic, and cardiovascular-protective effects (Robb and Stuart, 2014). Daidzein (25 μM) induced paraoxonase-1 (PON-1) activity and protected LDL from oxidation in Huh7 cells, thereby displaying an antiatherosclerotic potential (Schrader et al., 2012). It was known that PON-1 may regulate cholesterol efflux by stimulating the PPARγ-LXRα-ABCA1 pathway (Ikhlef et al., 2016), indicating that daidzein may regulate macrophage cholesterol efflux. Additionally, in another study utilizing cultured HepG2 cells, daidzein (EC₅₀ = 3.21 μM) upregulated the activity of CD36 and lysosomal integral membrane protein-II analogous-1 (CLA-1), which is a homolog of SR-BI, suggesting that daidzein may affect lipid uptake (Yang et al., 2009). Daidzein was also reported to be an inhibitor of HMG-CoA reductase, ACAT1, and ACAT2 (Borradaile et al., 2002; Sung et al., 2004).

In addition, in male middle-aged rats with HFD used to induce atherosclerosis, treatment with daidzein (30 mg/kg body weight) decreased the serum cholesterol and increased TG level (Sosić-Jurjević et al., 2007). In another study using rheumatoid arthritis rat model, it was shown that intragastric administration of daidzein (20 mg/kg body weight) and hesperidin (50 mg/kg body weight) reduced plasma VLDL, LDL-C, and TG concentrations, while increasing HDL-C levels (Ahmad et al., 2016).

7. Ellagic Acid

Ellagic acid, a dilactone of hexahydroxydiphenic acid, is an important antioxidant found in numerous fruits, such as cranberries, raspberries, grapes, pomegranates, as well as in walnuts. Ellagic acid has a wide range of biologic activities, including antiproliferative and antioxidative properties (Larrosa et al., 2010). One study utilizing J774A.1 murine macrophages suggested that ellagic acid (1 and 5 μM) stimulated cholesterol efflux by promoting the ABCA1 expression and upregulating PPARγ and LXRα levels (Park et al., 2011). This study also displayed that ellagic acid could modulate the SR-BI expression by counteracting PPARγ-responsive early signaling. Additionally, it was demonstrated that pomegranate ellagic acid (PEA) (10, 20, and 40 μg/ml) regulated the expression of PPARγ, ABCA1, and cholesterol 7α-hydroxylase (CYP7A1) in the hepatic cell line L-02, thus regulating cholesterol metabolism (Lv et al., 2016). Also, pomegranate peel polyphenols, especially PEA (25 and 50 μg/ml), inhibited macrophage lipid accumulation by decreasing the expression of CD36 and promoted ApoA1-mediated macrophage cholesterol efflux by upregulating ABCA1 and LXRα (Zhao et al., 2016).

In a hamster model with HFD, a high-dose of PEA (177 mg/kg body weight) decreased TC and TG in a dose-dependent manner and upregulated the expression of LXRα, PPARα, PPARγ, and their downstream gene ABCA1 (Liu et al., 2015). Moreover, using ApoE^−/− mice, it was shown that treatment of pomegranate phenolic compounds (200 μg/mouse per day) for 3 months, mainly including gallic acid and ellagic acid, reduced atherosclerotic lesions (Aviram et al., 2008).

8. Hesperetin

Hesperetin is an aglycone of hesperidin, a natural bioflavonoid that is present in Leguminosae, Dimorphicaceae, Labiatae, and Rutaceae plants. Hesperetin has many biologic and pharmacological activities, such as antioxidative, anti-inflammatory, and antiatherosclerotic effects (Yang et al., 2012; Ren et al., 2016). It was shown that hesperetin (5, 10, and 15 μM) reduced THP-1-derived foam cell formation by enhancing the expression of ABCA1 through increasing the activities of ABCA1 promoter and LXR enhancer, thus upregulating the ApoA1-mediated cholesterol efflux (Iio et al., 2012). Hesperetin (25 μM) may act as an antiatherogenic agent possibly by also inhibiting oxLDL-triggered ROS in human umbilical vein endothelial cells (HUVECs) (Choi et al., 2008), as well as the proliferation and migration of VSMCs (Wei et al., 2016). A recent study showed that hesperetin reduced plasma TC level, endothelial dysfunction, macrophage infiltration, and atherosclerotic lesion in ApoE^−/− mice (Sugasawa et al., 2019).

9. Icariin

Icariin (ICA) is present in the traditional Chinese herbal medicine Epimedium brevicornum Maxim. In recent years, ICA has been studied for its effect on CVD. It was shown that ICA possessed atheroprotective functions through various mechanisms, including counteracting endothelial dysfunction, suppressing the proliferation and migration of VSMCs, as well as inhibiting foam cell formation and inflammatory responses (Fang and Zhang, 2017). In models of normal rats (Hu et al., 2016b) or ApoE^−/− mice with HFD (Xiao et al., 2017), ICA (10–60 mg/kg body weight per day) significantly decreased the concentrations of TC, TG, and LDL-C. It was demonstrated that the inhibitory effects of ICA (4 μM) on cholesterol intake and foam cell formation were accompanied by a reduced expression of CD36 and an upregulated SR-BI expression through p38 MAPK pathway in THP-1 cells (Yang et al., 2015). Additionally, ICA (5 and 10 μM) lessened RAW264.7 macrophage infiltration at atherosclerosis lesion by blocking the CX3CR1-CX3CL1 interaction, which is highly related to monocyte adhesion and migration (Wang et al., 2016c).

10. Iris Isoflavones

Iris isoflavones (tectorigenin, irstectorigenins, and iristectorins) present in rhizomes of Iris germanica L. have many biologic activities, such as antioxidative, anti-inflammatory, and antiangiogenic effects (Jung et al., 2003). Some of these nature products could also regulate the metabolism of blood sugar and cholesterol (Lee et al., 2000a; Jung et al., 2002). It was reported that iristectorigenin B (5 and 10 μM) acted as a novel LXR modulator by regulating the transcriptional activity of LXRα/β in RAW264.7 cells. It also induced the activation of ABCA1 and ABCG1, thus increasing the macrophage cholesterol efflux (Jun et al., 2012).

11. Pratensein

Pratensein is an isoflavone, which is present in Trifolium pretense L. (red clover). It was reported that pratensein (10 μM) upregulated ABCA1 protein expression to increase the HDL levels in HepG2 cells (Gao et al., 2008). In the same line, another study indicated that pratensein (EC₅₀ = 1.08 μM) upregulated the CLA-1 expression, which is a human homolog of SR-BI, suggesting that it can play a potential role in the process of cholesterol efflux in vitro (Yang et al., 2007, 2009).

12. Puerarin

Puerarin is present in the roots of Pueraria (Radix puerariae). Due to positive action on dilating coronary artery, protecting ischemic myocardium, resisting myocardial ischemia and re-injury, and preventing atherosclerosis (Bao et al., 2015), puerarin has been extensively studied for the treatment of CVD. It is demonstrated that puerarin (25, 50, and 100 μg/ml) decreased the cellular lipid accumulation in THP-1 macrophages by promoting ABCA1-mediated cholesterol efflux through pathways involving miRNA-7, serine/threonine kinase 11 (STK11), and the AMP-activated protein kinase (AMPK)-PPARγ-LXRα-ABCA1 cascade (Li et al., 2017a). In the same model, puerarin (50 and 100 μg/ml) suppressed lipid deposition and foam cell formation by downregulating the expression of CD36 (She et al., 2014). It (10, 50, and 100 μg/ml) also suppressed oxLDL-induced macrophage activation and release of TNF-α and IL1-β by inhibiting the TLR4/NF-κB pathway. Furthermore, treatment with puerarin (140 and 200 mg/kg body weight per day) decreased the level of blood glucose, TC, TG, LDL-C, and increased the HDL-C level in the streptozotocin-induced diabetic rat model (Smith et al., 2006).

13. Quercetin

Quercetin, a polyhydroxy flavonoid, is one of the most abundant natural polyphenols in different foods, such as onions, apples, broccoli, and ginkgo. It has broad biologic activities, including anti-inflammatory, immunomodulatory, and cardiovascular-protective effects (Rauf et al., 2018). Some studies indicated that quercetin could interfere with foam cell formation (Lara-Guzman et al., 2012). Quercetin (50, 100, and 200 μM) enhanced ApoA1-mediated cholesterol efflux, as well as induced ABCA1 expression and the expression of PPARγ through activating the PPARγ signaling in THP-1-derived foam cells (Sun et al., 2015). Another study indicated that quercetin-increased ABCA1 expression possibly due to the p38-dependent pathway (Chang et al., 2012). It was also shown that quercetin-driven ABCA1 upregulation could be mediated by enhancing LXRα activity (Lee et al., 2013). Interestingly, a quercetin metabolite, quercetin-3-glucuronide, inhibited the formation of foam cells at 20 μM by suppressing the expression of SR-A1 and CD36 in RAW264.7 cells (Kawai et al., 2008). Moreover, quercetin was reported to inhibit atherosclerosis by promoting ABCA1- and ABCG1-dependent RCT in ApoE^−/− mice (Cui et al., 2017).

14. Silymarin

Silymarin, a mixture of flavonolignans from the medicinal plant Silybum marianum, includes silybin (synonymous with silibinin), isosilybin, silydianin, silychristin, and little amounts of other phenolic compounds. Within the last decade, several studies have suggested that, in addition to its use in the treatment of liver diseases, silymarin also has a protective effect on CVD. It was shown that four compounds from silymarin (isosilybin A, silybin B, silychristin, and isosilychristin) induced the expression of ABCA1 protein in THP-1 cells. Especially isosilybin A (10 and 30 μM) promoted cholesterol efflux from THP-1 macrophages due to its PPARγ-activating properties (Wang et al., 2015a). It is also reported that silymarin not only reduced the LDL and cholesterol levels, but also protected endothelial cells (Skottova and Krecman, 1998).

Furthermore, in the hypercholesterolemic rat model, supplementation of silybin (300 and 600 mg/kg body weight per day) in food significantly decreased serum levels of TC, TG, VLDL-C, LDL-C, and increased HDL-C, as well as reduced the formation of atherosclerotic plaques (Gobalakrishnan et al., 2016).

15. Wogonin

Wogonin, an O-methylated flavone, is present in the roots of Scutellaria baicalensis Georgi and the rhizomes of Scutellaria barbata L. It was shown that wogonin is effective in the treatment of inflammation, cancer, and anxiety (Tai et al., 2005; Li-Weber, 2009). Wogonin (40 μM) attenuated oxLDL-induced cholesterol accumulation in murine J774.A1 macrophages by enhancing cholesterol efflux through increasing the ABCA1 protein expression (Chen et al., 2011). In addition, this flavonoid decreased phosphorylated levels of ABCA1 protein. Another beneficial effect of wogonin in the context of prevention and therapy of atherosclerosis is to protect physiologic function in vascular endothelial cells and VSMCs (Oche et al., 2016). An in vivo study suggests that wogonin ameliorated hyperglycemia and dyslipidemia in db/db mice via PPARα activation (Bak et al., 2014).

1. Carotenoids

Carotenoids, also called tetraterpenoids, are a complex group of organic pigments with a basic tetraterpene skeleton and a variety of biochemical functions. A number of carotenoids have been reported to be associated with a wide range of bioactivities and potential health benefits (Rao and Rao, 2007; Fiedor and Burda, 2014). Epidemiologic studies, such as research regarding the association between β-carotene and CVD and case-control trails testing the concentration of β-carotene of patients with different CVD events, have demonstrated that the consumption of carotenoids can reduce the risk of CVD (Kohlmeier and Hastings, 1995; Riccioni et al., 2012). Here, the published studies regarding the effect of several main carotenoids, i.e., astaxanthin, β-carotene, capsanthin, retinoids, and lycopene, on cholesterol efflux and foam cells formation are discussed.

2. Ursolic Acid

Ursolic acid (UA), a natural ursane-type pentacyclic triterpenoid, is abundantly distributed in the plant kingdom. Recently, one study showed that UA (10 μM) promoted cholesterol flux from LDL-loaded macrophages to ApoA-1 (not HDL) through autophagy, without altering mRNA or protein levels of ABCA1 and ABCG1 in MPMs (Leng et al., 2016). Furthermore, UA (50 mg/kg) treatment in LDLR^−/− mice significantly reduced atherosclerotic lesion size, along with increase of macrophage autophagy (Leng et al., 2016). Taken together, these results provided evidence that UA may have potential to be further studied as an antiatherogenic agent.

3. Betulinic Acid

Betulinic acid, which is derived from the bark of yellow and white birch trees, is a pentacyclic triterpenoid with a wide range of pharmacological properties. One study indicated that betulinic acid may induce cholesterol efflux through blocking the NF-κB-miRNA-33s-ABCA1 signal pathway in LPS-treated macrophages (Zhao et al., 2013a). Consistently, in both RAW264.7 and THP-1 cells, betulin (a betulinic acid derivative) significantly increased ABCA1/ABCG1-mediated cholesterol efflux via suppressing the expression of SREBPs, which bind to E-box motifs in the ABCA1 promoter (Gui et al., 2016). Betulinic acid was reported to be a potent pharmacological inhibitor of ACAT1 and ACAT2 (Lee et al., 2006), suggesting its potential utility in reduced CE accumulation. Moreover, an in vivo study was done using ApoE^–/– mice in which increased ABCA1 expression and enhanced fecal cholesterol excretion were observed upon long-term administration of betulin (40 mg/kg), along with suppressed macrophage-positive areas in the aortic sinuses (Gui et al., 2016). Another study confirmed that betulinic acid (50 mg/kg) reduced atherosclerotic lesions and reduced TG, TC, and LDL-C levels in ApoE^−/− mice (Zhao et al., 2013a).

4. Erythrodiol

Erythrodiol is a pentacyclic triterpenoid present in olive oil. Recently, a study was conducted in THP-1 macrophages to screen the effect of various components of olive oil on foam cell formation. The results showed that among several compounds, only erythrodiol (10 μM) has a positive effect on ApoA-1-mediated cholesterol efflux by inhibiting ABCA1 degradation (Wang et al., 2017f). Therefore, erythrodiol may serve as a good candidate for further studies related to the prevention of atherosclerosis progression.

5. Ginsenosides

Ginseng, the original sources of which are the roots of the plant species Panax ginseng, is therapeutically used throughout the world for its possible curative and health-restorative properties. Most of the pharmacological actions of ginseng are attributed to its major constituents, ginsenosides. There are several types of ginsenosides, including Rb1, Rg1, Rg3, Rh1, Re, and Rd. Numerous studies focused on the potential benefits of the ginseng extract and its purified ginsenoside monomers in the area of CVD (Lee and Kim, 2014). Among these various ginsenosides, ginsenosides Rb1 and Rg1 are regarded as the most abundant active components from ginseng. A study showed that ginsenoside Rb1 (10 μM) significantly increased cholesterol efflux from foam cells to ApoA-1 by about 21% (Wang et al., 2007b). Similarly, a recent publication indicated that Rb1 (10–80 μM) treatment showed a significant increase in ABCA1 protein expression in macrophage foam cells, which provided the indirect evidence for a possible effect on the cholesterol efflux. Rb1 treatment also reduced the lipid metabolism and enhanced atherosclerotic plaque stability via enhancing macrophage autophagy in primary peritoneal macrophages isolated from C57BL/6 mice and ApoE^–/– mice (Qiao et al., 2017).

Ginsenoside Rd is another important constituent of ginseng. Data obtained from a RAW264.7 cell model revealed that Rd (20 μM) inhibited SR-A protein expression, followed by the decrease of oxLDL uptake and decreased intracellular cholesterol content, which is probably mediated by the inhibition of Ca²⁺ influx through Ca²⁺ channels (Li et al., 2011). Consistently, an in vivo study indicated that Rd treatment (20 mg/kg per day) reduced the oxLDL uptake and atherosclerotic plaque areas in ApoE^–/– mice.

6. Saikosaponin A

Saikosaponin A, a triterpenoid glycoside (saponin), is the major bioactive constituent from the Chinese herb Radix bupleuri. Saikosaponin A (at concentrations of 0–50 µM) ameliorated oxLDL-induced foam cell formation and suppressed lipoprotein uptake by diminishing LOX-1 and CD36 expression, as well as stimulated cholesterol efflux through upregulating the ABCA1 and PPARγ expression (He et al., 2016a). Saikosaponin A also regulated the immune inflammatory reaction via PI3K/Akt/NF-κB/NLRP3 signaling pathway and decreased the diffusion of proinflammatory cytokines (IL-1β, IL-18, IL-6, TNF-α, and MCP-1), revealing new insight on the potential of saikosaponin A in the context of atherosclerosis (He et al., 2016a).

7. Tanshinone IIA

Danshen, the rhizome of Salvia miltiorrhiza Bunge, has been widely used to treat various diseases for centuries (Gao et al., 2012). The chemical constituents and biologic activities of Danshen have been extensively studied. Among its constituents, tanshinone IIA is the major bioactive lipophilic compound (Xu and Liu, 2013; Fang et al., 2018). An in vitro study demonstrated that tanshinone IIA (at concentrations of 0.1–10 μM) decreased cellular cholesterol level, oxLDL uptake, as well as CD36 expression at both the mRNA and protein levels in oxLDL-treated MPMs (Tang et al., 2011). Furthermore, tanshinone IIA (1–10 μM) induced the reduction of CD36, which might be related to a PPARγ antagonism (Tang et al., 2011). Tanshinone IIA also increased the ABCA1- and ABCG1-mediated cholesterol efflux through the ERK/Nrf2/HO-1 loop and decreased the SR-A-mediated oxLDL uptake by inhibition of AP-1, resulting in decreasing cholesterol accumulation in cells (Liu et al., 2014c).

In vivo, tanshinone IIA (at doses of 10–90 mg/kg) also downregulated the SR-A mRNA expression and ameliorated the atherosclerotic lesions in the aortas of ApoE^−/− mice (Tang et al., 2011). Consistently, another study also showed that tanshinone IIA (30 mg/kg per day) lessened atherosclerotic plaques in ApoE^−/− mice (Liu et al., 2014c). Recently, another study conducted in macrophages from the peritoneal cavity of rats and THP-1-derived macrophages showed that ABCA1 mRNA and protein expression were significantly increased, while CD36 was significantly reduced upon tanshinone IIA treatment, thereby demonstrating simultaneous effects on cholesterol intake and efflux (Jia et al., 2016). In addition, tanshinone IIA was also reported to upregulate LDLR in hyperlipidemic rats (Jia et al., 2016). Furthermore, a clinical trial has shown that tanshinone IIA reduces hs-CRP in patients with CAD (Li et al., 2017b).

8. Tanshindiol C

Tanshindiol C is another bioactive compound isolated from Danshen. A recent report showed that tanshindiol C (1, 3, and 10 μM) concentration dependently inhibits oxLDL-induced foam cell formation via activation of Prdx1/ABCA1 signaling pathway. Also, tanshindiol C treatment-induced Prdx1 transcription was coregulated by Nrf2 and Sirt1 expression (Yang et al., 2018b). Furthermore, tanshindiol C also significantly inhibited the secretion of TNF-α, IL-1β, and IL-8 (Yang et al., 2018b). These data indicated that the potential therapeutic effects of tanshindiol C on atherosclerosis could be due to the modulation of both foam cell formation and inflammation. However, more conclusive evidence is needed to confirm the significance of tanshindiol C in the context of atherosclerosis in animal models and clinical trials.

9. Zerumbone

Zingiber zerumbet is a wild ginger commonly found in Asia. It has been used as a traditional herbal medicine to treat various diseases for a long time. A major bioactive component isolated from Zingiber zerumbet is zerumbone, which is a natural cyclic sesquiterpene known to possess numerous biologic activities. Zerumbone was reported to have therapeutic potential in the context of atherosclerosis. In vitro experiments showed that zerumbone (5 and 10 μM) suppressed the SR-A and CD36 mRNA expression via regulating AP-1 and NF-κB repression, leading to a blockade of acLDL uptake in THP-1 macrophages (Eguchi et al., 2007). Furthermore, THP-1 macrophages treated with zerumbone (10–100 μM) showed a significant reduction in the cholesterol levels via upregulation of mRNA and protein levels of ABCA1, but not ABCG1, coupled with the enhanced phosphorylation of ERK1/2 (Zhu and Liu, 2015). The consensus regarding the beneficial effects of zerumbone is strongly supported by experiments conducting in a cholesterol-fed rabbit model, which displayed that zerumbone (8–20 mg/kg) could prevent the development of atherosclerotic lesions (Hemn et al., 2013, 2015).

1. Gallotannin

1,2,3,4,6-Penta-O-galloyl-β-d-glucose (PGG) is a prodrug of gallotannin, which is present in diverse medicinal herbs. Zhao et al. (2015) found that PGG (2.5 and 5 μM) induced cholesterol efflux in oxLDL-stimulated J774A.1 and THP-1 macrophages by increasing SR-BI/ABCA1 expression, indicating that PGG has the potential to be further studied for promoting RCT and conferring atheroprotective effects.

2. Curcumin

Curcumin is a potent antioxidant present in Curcuma longa (turmeric or Jiang Huang) that has been demonstrated to confer protection against atherosclerosis in ApoE^−/− (Zhao et al., 2012) and LDLR^−/− mice (Hasan et al., 2014). It has been shown that curcumin and other bioactive phenolic compounds from turmeric have powerful bioactivities, including antioxidation associated with preventing lipid peroxidation (Ramirez Bosca et al., 1997). Curcumin was also used as a tool compound for inhibiting histone methyltransferase p300, c-Jun N-terminal kinases (JNK), and transcriptional factor AP-1 (Li et al., 2004b; Lee et al., 2009b; Huwait et al., 2011). By modifying AP-1, curcumin inhibited the resistin-induced dysregulation of SR-A, CD36, and ABCA1 (Lee et al., 2009b). In 2012, Zhao et al. (2012) systematically analyzed the effects of curcumin on macrophage-derived foam cell formation. The authors observed that curcumin (20 and 40 μM) significantly ameliorated lipid accumulation in J774.A1 macrophages induced by oxLDL by both decreasing the SR-A-dependent oxLDL uptake and increasing the ABCA1-dependent cholesterol efflux, without significant effect on other cholesterol transporters. Mechanistic experiments indicate that curcumin reduces SR-A expression via a ubiquitin/proteasome pathway. Furthermore, curcumin upregulated ABCA1 expression via LXRα-dependent transcriptional regulation (Zhao et al., 2012). Additional mechanisms, such as an activation of the AMPK/Sirt1/LXRα (Lin et al., 2015c) and the Nrf2/HO-1 pathway (Kou et al., 2013) and inhibition of the p38MAPK pathway (Min et al., 2013) were also involved in its effects. These concerted mechanisms together contribute to the inhibitory effect on foam cell formation mediated by curcumin. Recently, several curcumin derivatives, such as demethoxycurcumin (50 μM) (Kou et al., 2013) and nicotinate-curcumin (10 μM) (Gu et al., 2016), have also been shown to inhibit THP-1 macrophage-derived foam cell formation. In addition, curcumin functions as an inhibitor of PCSK9 and upregulates hepatic LDLR expression (Tai et al., 2014).

More importantly, curcumin (500–1000 mg/kg diet) attenuated the atherosclerosis development in ApoE^−/− mice by reducing the SR-A expression and increasing the ABCA1 expression in vivo (Hasan et al., 2014). The inhibitory activity against foam cell formation and the antiatherosclerotic effects of curcumin were also confirmed by several other studies (Kou et al., 2013; Min et al., 2013; Lin et al., 2015c; Soltani et al., 2017). Interestingly, curcumin lowered the level of LDL-C and TG in patients at risk for CVD (Qin et al., 2017). Therapeutic efficacy and molecular targets of curcumin in cardiovascular diseases have been reviewed elsewhere (Li et al., 2019).

3. Danshensu

Danshensu is an active component isolated from an eminent Chinese medicinal herb Salvia miltiorrhiza (Danshen), which is known for improving blood microcirculation. Danshen is widely used in China and other countries for treating various cardiovascular disorders, including atherosclerosis, angina pectoris, and myocardial infarction, due to potent anti-inflammatory, antioxidative, and lipid-lowering effects (Xu and Liu, 2013). It was shown that danshensu suppressed the LPS- and oxLDL-induced lipid accumulation in RAW 264.7 macrophages at the concentrations of 5–20 μM by activating LXR and inhibiting NF-κB, as well as the LPS-induced expression of fatty acid binding protein 4 and perilipin-2 (Xie et al., 2011; Wang et al., 2017c). Further studies also showed that danshensu (1–10 μM) prevented cholesterol accumulation and foam cell formation in RAW264.7 mouse macrophages by inhibiting CD36-mediated lipid uptake, while enhancing ABCA1 (G1)-mediated cholesterol efflux (Wang et al., 2010b; Gao et al., 2016). In addition, danshensu could improve dyslipidemia by decreasing the levels of LDL-C and fatty acid by inhibiting HMG-CoA reductase and fatty acid synthase expression (Yang et al., 2011).

An earlier in vivo study has revealed that danshensu (67.5 mg/kg body weight per day) attenuated high methionine-rich diet-induced accumulation of foam cells in rat aortic endothelium by suppressing the expression of TNF-α and intercellular adhesion molecule 1 (ICAM1) (Yang et al., 2010). Furthermore, Danshensu Bingpian Zhi (20 and 40 mg/kg body weight), which has the main bioactive constituent Danshensu, attenuated atherosclerosis in ApoE^−/− mice.

4. 6-Dihydroparadol

Ginger (Zingiber officinale) is a common food-derived health supplement, used worldwide due to its potential preventive effects in multiple diseases, including atherosclerosis and other inflammatory disorders (Aktan et al., 2006). Gingerols are the main bioactive compounds from ginger with potent anti-inflammatory properties in cultured cells. 6-Dihydroparadol, a bioactive metabolite of gingerols, displayed potent anti-inflammatory effects by blocking the NF-κB/inducible NO synthase/NO pathway in LPS-stimulated macrophages (Aktan et al., 2006). 6-Dihydroparadol (30 μM) was recently shown to promote cholesterol efflux from human THP-1 macrophages by increasing the expression of ABCA1 and ABCG1 via preventing the proteasome-dependent protein degradation, underscoring its therapeutic potential in preventing foam cell formation and possibly atherosclerosis (Wang et al., 2018a).

5. Paeonol

Paeonol is a bioactive phenolic compound isolated from traditional Chinese medicine Paeonia suffruticosa (Cortex Moutan), which is commonly used to treat inflammatory disorders including atherosclerosis (Zhao et al., 2013b). The antiatherosclerotic effects of paeonol have been well documented in several experimental animal models of atherosclerosis, including rabbits (Shi et al., 1988; Li et al., 2009a), quails (Dai et al., 1999), and ApoE^−/− mice (Zhao et al., 2013b; Li et al., 2015c). Four major antiatherosclerotic mechanisms of paeonol include 1) endothelial protection (reducing monocyte adhesion to inflamed endothelium, endothelial apoptosis, and senescence) (Nizamutdinova et al., 2007; Pan and Dai, 2009; Wang et al., 2012d; Bao et al., 2013; Chen et al., 2013; Jamal et al., 2014; Liu et al., 2014b; Yuan et al., 2016); 2) inhibiting the proliferation and migration of VSMC (Chen et al., 2014; Hu et al., 2016a); 3) antiplatelet activation (Shi et al., 1988); and 4) anti-foam cell formation (Zhao et al., 2013b; Li et al., 2015c).

Studies performed in isolated macrophages from ApoE^−/− mice treated with paeonol (50 and 100 μg/ml) showed that it significantly reduced the oxLDL-induced lipid accumulation, probably by promoting cholesterol efflux via activating the LXRα/ABCA1 pathway, which was suggested by study in J774.A1 macrophages (Zhao et al., 2013b). Silencing ABCA1 or LXRα abolished the paeonol-induced effects on cholesterol efflux and lipid accumulation, supporting the indispensable effect of LXRα and ABCA1 in mediating the protective effects. The inhibition of foam cell formation and antiatherosclerotic effects of paeonol were reproduced by another independent group, who confirmed the ABCA1-activating effects of this compound (5, 10 and 50 μM) in RAW264.7 macrophages and suggested that the anti-foam cell formation effects of paeonol also depended on CD36 inhibition and activation of HO-1 (Li et al., 2015c).

More importantly, paeonol treatment led to reduced atherosclerotic lesions formation and attenuated systemic inflammation, as well as increased ABCA1 expression in ApoE^−/− mice (Zhao et al., 2013b). In addition to effects mentioned above, paeonol also reduced the levels of malondialdehyde and oxidized LDL in hyperlipidemia rats (Dai et al., 2000).

6. Polydatin

Polydatin represents one of the major bioactive ingredients from Rhizoma Polygoni Cuspidati (Da Huang), an eminent Chinese medicinal herb, which dispels dampness, alleviates jaundice, reduces fever, subsides toxins, and removes stasis (Du et al., 2009; Deng et al., 2011; Liu et al., 2012b; Ma et al., 2016; Gugliandolo et al., 2017). Pharmacological studies in the past decade have shown that polydatin exhibits a broad range of bioactivities with cardiovascular relevance, including antioxidation, anti-inflammation, cardioprotection, blood vessel dilation, lipid-lowering effect, as well as inhibitory effects on platelet aggregation, monocyte adhesion to activated endothelium, thrombus formation, and atherosclerosis development (Du et al., 2009; Deng et al., 2011; Liu et al., 2012b; Ma et al., 2016; Gugliandolo et al., 2017). The preventive effects of polydatin on foam cell formation were reported recently (Wu et al., 2015b). Polydatin treatment of 48 hours reduced the level of TC, FC, and CE, together with reducing the secretion of TNF-α and IL-1β in oxLDL-stimulated ApoE^−/− mouse macrophages. The mechanism of these effects is linked to the activation of PPARγ-dependent ABCA1 upregulation and the decrease of CD36 expression (Wu et al., 2015b). Polydatin also improved dyslipidemia via suppressing PCSK9 and upregulating hepatic LDLR expression (Li et al., 2018a).

7. Protocatechuic Acid

Protocatechuic acid (PCA) is a bioactive compound present in some medicinal herbs such as Danshen (Li et al., 2018d)). It is a major metabolite of fruit/vegetable-derived anthocyanins (such as C3G) generated by gut microflora (Hidalgo et al., 2012; Wang et al., 2012a; Lin et al., 2015b). Consumption of the PCA-rich vegetable chicory (5 g/kg diet) reduced the TC and CE accumulation in isolated macrophages via upregulation of the ABCA1/ABCG1-mediated cholesterol efflux (Lin et al., 2015b). Because of the fact that PCA is the major component of chicory, a later study found that PCA, rather than its parent drug C3G, promoted the ABCA1 (G1)-dependent cholesterol efflux from macrophages. miRNA microarray assay elucidated that PCA reduced the miRNA-10b expression, thereby upregulating the expression of ABCA1 (G1) in MPMs (Wang et al., 2012a). Protocatechuic acid could also regulate lipid metabolism via suppressing the expression of HMG-CoA reductase (Liu et al., 2010b). In in vivo study, C3G (50 mg/kg body weight) induced RCT in the ApoE^−/− mice model and the atheroregression could be inhibited by antibiotic treatment, suggesting that the atheroprotective effect of C3G is gut microbiota dependent (Wang et al., 2012a).

In addition to influence of cholesterol metabolism, PCA (10, 20, and 40 μg/ml) was also shown to inhibit ICAM1 and vascular cell adhesion molecule 1 (VCAM-1)-dependent monocyte adhesion to activated HUVECs, as well as CCL2-mediated monocyte transmigration, thereby reducing the development of atherosclerosis in ApoE^−/− mice at the dose of 0.03 g/kg (Wang et al., 2010a, 2011; Stumpf et al., 2013). PCA (150 μg/ml) also exhibited inhibitory effects on VSMC proliferation in A7r5 smooth muscle cell line (induced by oleic acid) by activating AMPK and arresting cell cycle at G₀/G₁ phase (Lin et al., 2015a). Also, PCA increased the endothelium-dependent vasorelaxation as well as tetrahydrobiopterin levels and combated eNOS uncoupling (Liu et al., 2016b).

8. Salicylic Acid

Aspirin (acetyl salicylic acid) is an anti-inflammatory drug used as a cardiovascular therapeutic. It (600 and 1200 μM) upregulated the expression of ABCA1 and SR-BI, thereby stimulating the HDL-mediated cholesterol efflux in THP-1 macrophages (Viñals et al., 2005; Lu et al., 2010). In light of the key role of AMPK in regulating lipid and energy metabolism, Fullerton et al. (2015) explored whether AMPK was involved in the induction of ABCA1 and ABCG1 expression by aspirin. The authors observed that AMPK β1 deletion significantly impaired cholesterol efflux, without affecting lipid uptake in macrophages. Moreover, the AMPK activation by salicylate (salts and esters of salicylic acid) prevented foam cell formation via promoting HDL and ApoA-1-mediated cholesterol efflux by increasing the ABCA1 and ABCG1 expression in BMDMs. However, the preventive effect of salicylate was reduced in AMPK β1-deficient BMDMs, indicating that the effect of salicylate on cholesterol efflux and foam cell formation is dependent on AMPK (Fullerton et al., 2015). Another study confirmed this result and observed that a low-dose of aspirin (<0.5 mM) also upregulates the ABCA1-dependent cholesterol efflux via activating the PPARα pathway, which provides additional insights to explain the cardiovascular actions of aspirin (Wang et al., 2010e). In addition, aspirin attenuated atherosclerosis in ApoE^−/− mice partly by suppressing systemic inflammation and promoting inflammation resolution (Petri et al., 2017).

9. Salvianolic Acid B

Salvianolic acid B is a major hydrophilic constituent from Danshen, which has been demonstrated to exhibit antiatherosclerotic effects in a model of neointimal hyperplasia in rabbits (Yang et al., 2011) and in ApoE^−/− mice (Chen et al., 2006; Lin et al., 2007). Salvianolic acid B has shown to inhibit lipid peroxidation (LDL oxidation) (Yang et al., 2011) and activate AMPK (Cho et al., 2008). A high-throughput screening assay identified salvianolic acid B (15 and 30 µM) as an effective CD36 antagonist that blocks the oxLDL uptake in RAW264.7 macrophages (Wang et al., 2010b). Further follow up studies showed that salvianolic acid B directly bound to CD36 (K_d = 3.74 μM) and inhibited the CD36 expression. By doing so, salvianolic acid B (1, 10, and 100 µM) reduced the CD36-dependent lipid uptake and foam cell formation in mouse macrophages, as well as in PMA-stimulated THP-1 human macrophages (Bao et al., 2012). Salvianolic acid B has been reported to promote the HDL- and ApoA-1-mediated cholesterol efflux in differentiated THP-1 macrophages via inducing the expression of ABCA1 (Yue et al., 2015). Further mechanistic studies showed that the upregulation of ABCA1, promoted by salvianolic acid B (1 and 10 µM), was reversed by PPARγ and LXRα inhibitors. This evidence indicates that salvianolic acid B reduces lipid accumulation by promoting cholesterol efflux via a PPARγ/LXRα/ABCA1-dependent pathway in THP-1 macrophages (Yue et al., 2015).

10. Sesamol

Sesamol is an essential bioactive compound of sesame oil (Sesamum indicum), which is a cardiovascular protective dietary supplement. Previous studies have shown that a sesame oil aqueous extract (0.75 mg/mouse per day) prevented or regressed atherosclerosis in LDLR^−/− mice (Narasimhulu et al., 2018) and sesamol derivative (INV-403) (20 mg/kg per day) played a positive role in hyperlipidemic rabbits fed with an atherogenic diet (Ying et al., 2011). Mechanistic studies showed that both sesame oil and sesame oil aqueous extract slightly increased the ABCA1 expression, while decreasing the mRNA expression of CD36, CD68, SR-A, and LOX-1 in the aortic arch segments of LDLR^−/− mice model (Narasimhulu et al., 2018).

Furthermore, it is possible that sesamol is mainly responsible for the observed atheroprotective effects of sesame oil. A recent study identified that both sesamol (25–100 µM) and sesame oil (1–10 μg/ml) increase the expression/activity of PPARγ and LXRα in Chinese hamster ovary cells via an MAPK-dependent mechanism. Most importantly, sesamol and sesame oil boosted cholesterol efflux from MPM (Majdalawieh and Ro, 2015). This evidence, together with a previous report showing that sesamol (10, 30, and 100 µM) suppressed inflammatory response in LPS-stimulated RAW264.7 mouse macrophages (via AMPK-dependent NF-κB suppression) (Wu et al., 2015d), collectively indicates that sesamol has the potential to reduce foam cell formation and atherogenesis (Majdalawieh and Ro, 2015).

11. Resveratrol

Resveratrol is a major stilbenoid compound isolated from red wine. Resveratrol displays reproducible antiatherosclerotic effects in several animal models, including ApoE^−/− mice (Do et al., 2008; Chang et al., 2015), ApoE*3-Leiden CETP mice (Berbée et al., 2013), and ApoE^−/−/LDLR^−/− mice (Fukao et al., 2004). The atheroprotective mechanisms of resveratrol include inhibition of LDL oxidation (Berrougui et al., 2009), enhancement of endothelial protection, decrease of TMAO via gut microbiota (Chen et al., 2016), inhibition of the proliferation and migration of VSMCs, monocyte/macrophage differentiation, and platelet activation (Vasamsetti et al., 2016). It was reported that resveratrol (2.5 µM) reduced the LPS-induced RAW264.7 foam cell formation via attenuating the NADPH oxidase 1 (Nox1)-dependent ROS production and MCP1 expression via Akt/Foxo3a and AMPK/Sirt1 pathways (Park et al., 2009a; Dong et al., 2014). Follow up studies in human macrophages identified that resveratrol at the concentrations of 10 or 100 µM reduced oxLDL uptake in THP-1 macrophages (Voloshyna et al., 2013). Resveratrol promoted ApoA1- and HDL-mediated cholesterol efflux in both mouse (RAW264.7, J774.A1, MPMs) and human macrophages (THP-1) by increasing the expression of ABCA1 (Berrougui et al., 2009; Allen and Graham, 2012) and ABCG1 via PPARv/LXRα and adenosine 2A receptor pathway (Voloshyna et al., 2013). In addition to the therapeutic effects in foam cell formation induced by oxLDL, resveratrol (10 µM) also ameliorated foam cell formation in J774.A1 macrophages caused by Chlamydia pneumonia infection by inhibiting the synthesis of IL-17A (Di Pietro et al., 2013). Clinical studies have shown that resveratrol lowers the level of TC and TG in patients with dyslipidemia (Simental-Mendia and Guerrero-Romero, 2019). Resveratrol also regulated lipid metabolism via inhibiting cholesterol-ester-transport protein and HMG-CoA expression/level (Cho et al., 2008).

12. Epigallocatechin Gallate

EGCG is the most studied polyphenol (catechin), derived from tea and possessing antiatherosclerotic and plaque-stabilizing effects in rats, rabbits, and ApoE^−/− mice (Chyu et al., 2004; Xu et al., 2014a; Wang et al., 2018e,f). Previous studies have shown that EGCG (25 µM) attenuated the oxLDL-induced apoptosis of HUVECs by blocking JNK activation (Choi et al., 2008). Furthermore, EGCG (40 and 80 µg/ml) reversed the TNF-α-induced ABCA1 downregulation and reduced the cholesterol efflux from THP-1 macrophages by activating the Nrf2-dependent NF-κB inhibitory effects (Jiang et al., 2012). In the same cell line, EGCG (10 μM) also blocked the oxLDL-induced upregulation of SR-A, thus blocking the oxLDL uptake and foam cell formation (Chen et al., 2017). Mechanistic studies also showed that EGCG is having a preventing action on hyperlipidemia by increasing the expression and activity of LDLR (Lee et al., 2008).

1. Lignans

Lignans represent a class of natural polyphenols, which are dimers derived from two molecules of a phenylpropanoid derivative (a C6-C3 monomer). They are present in flaxseed, sesame, soybeans, and some fruits. By acting as phytoestrogens, dietary lignans exhibit potent antiviral, antioxidative, antitumor, and antiatherosclerotic activities (Peterson et al., 2010).

2. α-Asarone

As a major active constituent of Acorus tatarinowii Schott, α-asarone exhibits a wide range of bioactivities. It was observed that purple perilla extract with α-asarone increased the expression of ABCA1 and ABCG1 and accelerated the cholesterol efflux from lipid-loaded J774A.1 macrophages. It also enhanced the expression of LXRα and PPARγ in vitro (Park et al., 2015). These data indicated that α-asarone promoted the macrophage cholesterol efflux through the PPARγ-LXRα-ABC transporters pathway.

In terms of lipid metabolism, a previous study has shown that there is an association between the intake of α-asarone (80 mg/kg body weight per day) and the decreased level of serum cholesterol in hypercholesterolemic rats by inhibition of HMG-CoA reductase (Rodríguez-Páez et al., 2003). Another animal study revealed that 2,4,5-trimethoxycinnamic acid, the major and nontoxic metabolite of α-asarone, had most of the pharmacological properties of α-asarone. Both compounds lowered TC, LDL-C, and HDL-C in hypercholesterolemic rats (Antunez-Solis et al., 2009).

3. Chlorogenic Acid

Chlorogenic acid, a naturally occurring phenolic acid present in coffee, as well as in the leaves and fruits of diverse dicotyledonous plants, acts as an important intermediate of lignin biosynthesis. Chlorogenic acid exhibited various effects, like antioxidative activity and modulation of blood glucose and cholesterol metabolism (Rodriguez de Sotillo and Hadley, 2002; Tošović et al., 2017). In vivo, it reduced (at 200 and 400 mg/kg body weight per day) the percentage and the total atherosclerotic lesion area and aortic dilatation in cholesterol-rich diet-fed ApoE^−/− mice, as well as decreased levels of TC, LDL-C, and TG in serum (Wu et al., 2014).

Recent reports suggested that chlorogenic acid also displays a modulatory effect on foam cell formation. Chlorogenic acid (1 and 10 µM) inhibited foam cell formation and decreased the oxLDL-elicited neutral lipid and cholesterol accumulation in RAW264.7 macrophages via increasing the transcription of PPARγ, LXRα, ABCA1, and ABCG1 (Wu et al., 2014). Interestingly, the serum containing chlorogenic acid metabolites inhibited the oxLDL-induced lipid accumulation and increased the cholesterol efflux in RAW264.7 cells. Furthermore, five serum metabolites of chlorogenic acid were tested and the results showed that caffeic, ferulic, and gallic acids significantly increased the HDL-mediated cholesterol efflux from RAW264.7 cells (Wu et al., 2014). These data suggest that these metabolites might be potential bioactive compound, accounting for the in vivo effect of chlorogenic acid. In addition, it is likely that other mechanisms are involved in the lipid regulation by this natural product. In this line, it was reported that chlorogenic acid (30 µM) promoted the efflux of TC and triacylglycerol and increased mRNA expression of ABCA1, CYP7A1, and AMPKα2 in HepG2 cells (Hao et al., 2016).

4. Caffeic and Ferulic Acid

Caffeic acid is a well-known phenolic phytochemical present in coffee and diverse other plants, since it represents a key intermediate in the biosynthesis of lignin. The closely related ferulic acid is a hydroxycinnamic acid formed by the conversion of caffeic acid and it is also present in the cell walls of diverse plants. Both caffeic acid and ferulic acid exhibit anticancer, antioxidative, and diverse other biologic activities (Sato et al., 2011; Rosendahl et al., 2015). Both compounds are being extensively studied for their potential benefits in various disorders, such as inflammation, neurodegenerative diseases, cancer, CVDs, and atherosclerosis (Zhao and Moghadasian, 2008).

It was reported that caffeic and ferulic acid, the major phenolic acids of coffee, enhanced HDL-mediated cholesterol efflux, but not the ApoA-1-mediated efflux in THP-1 macrophages. Both compounds (1 µM) increased the expression of ABCG1 and SR-BI, but not ABCA1 (Uto-Kondo et al., 2010). Further experiments showed that caffeic acid was identified as a PPARα agonist in vitro (Kim et al., 2014). As a metabolite of chlorogenic acid, ferulic acid (0.25, 0.5, and 1 µM) has also been shown to have an enhancement effect on HDL-mediated cholesterol efflux from macrophages through increasing the expression of ABCG1 and SR-BI (Uto-Kondo et al., 2010). Furthermore, it was demonstrated that ferulic acid (1 µM) displays antiatherosclerotic potential by increasing the ABCA1 and ABCG1 expression in macrophage form cells and further promoting cholesterol efflux (Chen and Wang, 2015). In this light, counteraction of foam cell formation can be also considered in relation to the potential antiatherogenic properties of phenylpropanoids, in addition to their antioxidative/anti-inflammatory functions.

1. Arecoline

Arecoline is a nicotinic acid-based bioactive alkaloid isolated from areca nut, which has a medicinal potential in the treatment of neurodegenerative diseases (Ghelardini et al., 2001). Cardiovascular benefits of arecoline are largely unknown. In oxLDL-stimulated macrophages, arecoline reduced the cholesterol accumulation in a dose-dependent manner by reducing TC, FC, and CE. In addition, arecoline also promoted the cholesterol efflux by inducing ABCA1 expression. Further studies were warranted to evaluate the effects of arecoline on lipid uptake in RAW264.7 macrophage and its therapeutic effect on atherosclerosis in vivo (Ouyang et al., 2012). In vivo study showed that arecoline suppressed atherosclerosis in ApoE^−/− mice by inhibiting NF-κB activation (Zhou et al., 2014).

2. Berberine

Berberine is a bioactive alkaloid isolated from several medicinal plants, including Berberis (Huang Lian). Berberine-containing plants have been used in China for a long time to treat various disorders, including CVD (Feng et al., 2019). Berberine protects against atherosclerosis in various animal models due to its lipid-modulating effects. Two well-elucidated molecular targets of berberine in modulating lipid levels are LDLR (Kong et al., 2004) and proprotein convertase subtilisin/kexin type 9 (PCSK9) (Dong et al., 2015). In 2010, Lee et al. (2010) observed that berberine (5, 10, and 20 µM) inhibited THP-1 macrophage foam cell formation by promoting LXRα/ABCA1-dependent cholesterol efflux. However, berberine did not affect ABCG1, SR-BI, CD36, and SR-A. Further studies indicate that berberine (5 and 10 mg/l) also prevented the oxLDL-induced upregulation of LOX-1 and downregulation of SR-BI in THP-1 macrophages, without affecting the SR-A and ABCA1 expression (Guan et al., 2010; Chi et al., 2014a). In addition, several derivatives of berberine, such as 13-hexylberberine (Li et al., 2010), were shown to act as potential CD36 inhibitors. Possible inhibition of foam cell formation and antiatherosclerotic potential of these derivatives needs to be further evaluated in vitro and in vivo. Other mechanisms linked to the effect of berberine on foam cell formation include activation of AMPK/Sirt1 (Chi et al., 2014b), autophagy induction (Kou et al., 2017), as well as inhibition of adipocyte enhancer-binding protein 1 (Huang et al., 2012b).

However, a contradictory study reported that berberine (5 mg/kg per day) unexpectedly promoted atherosclerosis in mice by enhancing the SR-A-mediated oxLDL uptake and foam cell formation in human and mouse macrophages (Li et al., 2009b). Mechanistic studies revealed that berberine-mediated SR-A upregulation was exerted through suppressing the phosphatase and tensin homolog expression, thus promoting the activation of Akt in RAW264.7 macrophages (Li et al., 2009b). The discrepancy of the atherosclerosis-modulating effects of berberine may be due to different animal models and doses, which were used in these studies. Despite most of the literature indicating that berberine has beneficial effects in cardiovascular and metabolic diseases, the precise effects and mechanisms of berberine action in the modulation of foam cell formation need to be further evaluated. In patients with dyslipidemia, berberine reduces TC, LDL-C, TG and increases HDL-C, partially through inhibiting PCSK9 and increasing LDLR expression/activity (Kong et al., 2004; Cameron et al., 2008; Ju et al., 2018).

3. Piperine

Piperine is a biologically active ingredient isolated from the fruits of black pepper (Piper nigrum). It has been shown that piperine (100 μg/ml) decreases cholesterol uptake dose dependently in Caco-2 cells by reducing the levels of the membrane localized cholesterol transporter protein-Niemann-Pick disease, type C1 (NPC1) like intracellular cholesterol transporter 1 (NPC1L1) and SR-BI (Duangjai et al., 2013). More recently, piperine (25, 50, and 100 µM) was also found to promote the ABCA1 protein expression in THP-1-differentiated human macrophages, without affecting the ABCG1 and SR-BI expression (Wang et al., 2017d). Further studies revealed that piperine did not affect the gene expression of ABCA1, but increased the ABCA1 protein stability by preventing calpain-mediated ABCA1 protein degradation (Wang et al., 2017d). In addition, piperine regulated lipid metabolism via increasing hepatic LDLR expression via proteolytic activation of SREBPs in HepG2 cells (Ochiai et al., 2015). These pharmacological effects placed piperine as a promising food-derived bioactive compound with potential therapeutic implications in atherosclerosis.

4. Rutaecarpine

Rutaecarpine (or rutecarpine) is a nonbasic alkaloid isolated from the unripe fruits of the medicinal herb Evodia rutaecarpa (Wu Zhu Yu), which has been used in treating cardiovascular and cerebrovascular diseases (Tian et al., 2019b). A high-throughput screening assay for ABCA1 upregulators identified rutaecarpine as a potential active compound that increased the ABCA1 promotor activity in HepG2 cells (EC₅₀ = 0.27 μM) (Xu et al., 2014b). Further studies showed that rutaecarpine upregulated the expression of ABCA1 and SR-BI (without affecting ABCG1 and CD36) via LXRα and LXRβ, thereby reducing the lipid accumulation and foam cell formation via promoting cholesterol efflux in vitro (RAW264.7 macrophages and HepG2 cells) and in vivo (ApoE^−/− mouse). By this mechanism, rutaecarpine (20 mg/kg body weight per day) reduced atherosclerotic plaque development in ApoE^−/− mice. The atheroprotective effects of rutacarpine are accompanied by reduced macrophage and lipid content in atherosclerotic plaques (Xu et al., 2014b). Rutaecarpine lowered the level of TC, TG, LDL-C, and hs-CRP in hyperlipidemic and hyperglycemic rats via AMPK activation and NF-κB inhibition (Nie et al., 2016; Tian et al., 2019b). Several derivatives of rutaecarpine also exhibited antiatherosclerotic potential by enhancing cholesterol efflux from RAW264.7 macrophages to HDL and thus inhibiting foam cell formation (Li et al., 2014).

5. Evodiamine

Evodiamine, an indoloquinazoline alkaloid, is present in the traditional Chinese medicine Fructus Evodiae (Chinese name: Wuzhuyu) (Shoji et al., 1986; Wagner et al., 2011). Evodiamine (3–20 µM) increased cholesterol efflux from THP-1-derived human macrophages significantly by directly binding to ABCA1 and thereby increasing ABCA1 stability and protein level (Wang et al., 2018c). Moreover, treatment of evodiamine (10 mg/kg body weight) for 4 weeks decreased the size of atherosclerotic lesions and alleviated the hyperlipidemia, as well as hepatic macrovesicular steatosis in ApoE^−/− mice, probably through transient receptor potential vanilloid type 1 (TRPV1) pathway (Su et al., 2014).

6. Leonurine

Leonurine is an anti-inflammatory, antioxidative, and antiatherosclerotic pseudoalkaloid isolated from Herba leonuri (Jiang et al., 2017a). Recently, Jiang et al. (2017a) have demonstrated that leonurine (5–80 µM) dose dependently inhibited the lipid accumulation (TC, FC, and CE) and foam cell formation in oxLDL-stimulated THP-1 macrophages. Mechanistic investigations indicated that leonurine inhibited foam cell formation by promoting ApoA-1- and HDL-mediated cholesterol efflux via the PPARγ/LXRα/ABCA1 (G1) pathway (Jiang et al., 2017a). In this line, leonurine also increased the expression of key proteins in cholesterol efflux, including PPARγ, LXRα, ABCA1 (G1), and reduced atherosclerotic development in ApoE^−/− mice fed with an atherogenic diet (10 mg/kg body weight per day). More studies are warranted to study potential effects of leonurine on suppressing lipid uptake and the related mechanisms.

1. Diosgenin

Diosgenin and its glycoside form dioscin are pharmacologically active steroidal sapogenins present in Dioscorea plant species. Both diosgenin and dioscin have been shown to exhibit antioxidative, hypolipidemic, antithrombotic, and endothelial-protective effects (Son et al., 2007; Gong et al., 2011; Liu et al., 2012a; Lv et al., 2015; Wu et al., 2015c; Wang et al., 2017g). Due to their structural similarity to cholesterol, diosgenin and dioscin were used in industrial production of steroidal drugs and are known to be increasing cholesterol secretion as well as inhibiting cholesterol absorption (Son et al., 2007). Diosgenin displayed antiapoptotic effects against H₂O₂-induced apoptosis (Gong et al., 2010), as well as anti-inflammatory effects in VSMCs by inhibiting the MAPK/Akt/NF-κB pathway (Choi et al., 2010). Diosgenin (14 μM) also blocked atherosclerosis by inhibiting the nuclear translocation of notch intracellular domain in THP-1 cells (Binesh et al., 2018). It also inhibited the TNF-α-induced leukocyte adhesion to activated endothelial cells by inhibiting the upregulation of ICAM1, VCAM1, and endothelial lipase (Wu et al., 2015c). In addition, dioscin (1–4 μM) inhibited oxLDL uptake by blocking systemic inflammation and the LOX-1/NF-κB pathway in MPMs from atherosclerotic rats (Wang et al., 2017g). Also, diosgenin promoted cholesterol efflux by increasing the ABCA1 expression, independent of LXRα (Lv et al., 2015). The potential mechanism of diosgenin (10–80 μM) in reducing foam cell formation in THP-1 macrophages as well as in atherogenesis is proposed to be through inhibiting of miRNA-19b (Lv et al., 2015).

2. Fucosterol

Fucosterol is a sterol that is present in marine algae. It has beneficial effects in hypercholesterolemia, due to its activity to reduce LDL-C and increase HDL-C (Hoang et al., 2012). A recent study (Hoang et al., 2012) has shown that fucosterol is a dual LXRα/β agonist that promotes cholesterol efflux from THP-1 macrophages at 100 and 200 μM, by increasing the expression of efflux transporters-ABCA1, ABCG1, as well as ApoE; meanwhile, it also reduces the intestinal NPC1L1-mediated cholesterol absorption. This is a beneficial effect, as it can avoid the side effects associated with the LXR activation, probably due to upregulating Insig-2a, which is a negative regulator of the lipogenic transcription factor SREBP-1c (Hoang et al., 2012).

3. Panax Notoginseng Saponins

PNS are the major bioactive ingredients of the medicinal herb P. notoginseng (Duan et al., 2017). PNS have extensive cardiovascular protective effects, including preventing endothelial dysfunction and increasing blood flow, antioxidation, anti-inflammation, antithrombosis, inhibition of foam cell formation, and regulation of cardiac function (Jia et al., 2010; Yuan et al., 2011). PNS (40 and 80 mg/l) decreased the accumulation of cholesterol esters via increasing the ABCA1 expression in macrophages (Jia et al., 2010). In vivo study shows that PNS inhibited foam cell formation in zymosan A-induced atherosclerosis rats at the dose of 100 mg/kg per day (Yuan et al., 2011). Recently, ginsengoside Rd, a purified constituent from PNS, also demonstrated antiatherosclerotic effects in ApoE^−/− fed with atherogenic diet by attenuating foam cell formation (Li et al., 2011). Mechanistic studies indicated that ginsengoside Rd (20 μM) blocked SR-A-mediated oxLDL uptake via blocking voltage-independent Ca²⁺ channels in RAW264.7 cells (Li et al., 2011). It remains to be elucidated whether other saponins from PNS have protective effects against foam cell formation and may contribute to the atheroprotective effects of PNS.

4. Vitamin D

Vitamin D deficiency is associated with an increased risk of CVDs, such as hypertension and atherosclerosis (Weng et al., 2013). A landmark study in 2009 showed that deficiency of vitamin D promoted the modified LDL-induced foam cell formation of macrophages from patients with diabetes (Oh et al., 2009). Similarly, deficiency of macrophage vitamin D aggravated CD36 and SR-A-mediated lipid uptake (via JNK activation), as well as promoted insulin resistance to increase atherosclerosis in mice (Oh et al., 2015). Vitamin D deficiency-induced hypertension and atherosclerosis in LDLR^−/− mice were reversed by JNK2 deficiency (Oh et al., 2018). A recent study has shown that vitamin D deficiency decreased the HDL level and LXR/ABCA1(G1) expression, thus augmenting cholesterol accumulation and atherosclerosis in hypercholesterolemic microswine (Yin et al., 2015). On the other side, vitamin D supplementation inhibited the CD36 and SR-A-mediated lipid (oxLDL and ac-LDL) uptake and promoted the nascent HDL generation in HepG2 cells via ABCA1-dependent cholesterol efflux. The final outcome of these effects is to reduce foam cell formation in vitamin D-treated cells (Oh et al., 2009; Yin et al., 2015). Vitamin D supplementation improves glycemic control, increases HDL-C, and decreases hs-CRP levels in patients with CVD (Ostadmohammadi et al., 2019).

1. Docosahexaenoic Acid and Eicosapentaenoic Acid

Increasing evidence has shown that the consumption of fish oil and its main bioactive components EPA and DHA leads to a reduced risk of CVD. One major mechanism of the cardiovascular effects of fish oil is modulation of lipid homeostasis and foam cell formation (McLaren et al., 2011b). Studies observed that free polyunsaturated fatty acids buffer including DHA and EPA at 5 μM significantly reduced the CD36 expression in human U937 monocytes (Pietsch et al., 1995). Furthermore, DHA and EPA (25, 50, and 100 μM) inhibited acLDL uptake in human THP-1 macrophages partially through reduction of mRNA and protein expression of CD36 and SR-A. Other SR-independent mechanisms include reduction of macropinocytosis and expression of syndecan-4, which is involved in the uptake of other forms of modified LDL (McLaren et al., 2011b). Moreover, an earlier study in 1992 showed that EPA (100, 300 mg/kg body weight per day) inhibits the CE accumulation in macrophages by decreasing the expression of receptors of acLDL in Wistar rats (Saito et al., 1992). These studies underscore the preventive and therapeutic potential of EPA and DHA in atherosclerosis.

2. 13-Hydroxyoctadecadienoic Acid

13-Hydroxyoctadecadienoic acid (also known as 13-HODE, 13-hydroxy linoleic acid) is a major oxidized lipid component of oxLDL and a natural and endogenous PPAR agonist (Nagy et al., 1998; Kämmerer et al., 2011). Treatment with 13-hydroxyoctadecadienoic acid (1 and 2.5 μM), but not with linoleic acid, increased the transactivation activity of PPAR, and downstream targets of PPAR/LXRα pathway, including ABCA1 (G1) and SR-BI. By doing so, 13-HODE (2.5 μM) promoted ApoA-1-mediated cholesterol efflux in RAW264.7 macrophages, thus decreasing cellular cholesterol level and foam cell formation (Kämmerer et al., 2011).

3. Linoleic Acid

Dietary supplementation of isomers of conjugated linoleic acids has been known for a long time to promote the regression of pre-established atherosclerotic plaques (Mooney et al., 2012; Song et al., 2013). These compounds conferred atheroprotection mainly by functioning as endogenous activators of PPARα, PPARγ, and PPARG coactivator 1 alpha (PGC1α) (Ringseis et al., 2008). Conjugated linoleic acids isomers, such as c9t11-CLA (50 μM) and t10c12-CLA (50 μM), were shown to stimulate the ApoA-1-mediated cholesterol efflux, thus reducing lipid accumulation by increasing the expression of NPC-1, NPC-2, ABCA1, and LXRα (Ringseis et al., 2008; Reza et al., 2009). In addition, 1% conjugated linoleic acid blend (80:20 cis-9,trans-11-CLA:trans-10,cis-12-CLA) also induced the regression of pre-established atherosclerotic plaques in ApoE^−/− mice by promoting macrophage polarization toward a M2 anti-inflammatory phenotype (McCarthy et al., 2013). Published literature suggests that linoleic acid could possibly reduce TC via increasing the hepatic LDLR expression and activity (Ringseis et al., 2006).

1. l-(+)-Citrulline

In the NO cycle, l-(+)-citrulline is a side product of the eNOS-mediated NO production which uses l-arginine as a substrate. l-(+)-Citrulline reduces endothelial senescence, VSMC proliferation, and atherosclerosis in several animal models, partially through increasing the NO bioavailability (Ruiz et al., 1999; Hayashi et al., 2005, 2006; Morita et al., 2014; Tsuboi et al., 2018). Citrulline (1 mM) also increased the ABCA1 and ABCG1 expression in differentiated THP-1 macrophages, thereby promoting HDL- and ApoA-1-mediated cholesterol efflux (Uto-Kondo et al., 2014). Of clinical relevance, citrulline consumption (3.2 g/day for 1 week) increased the level of citrulline and arginine post citrulline sera and promoted the HDL- and ApoA-1-mediated cholesterol efflux by increasing the expression of both ABCA1 and ABCG1 in BMDM (Uto-Kondo et al., 2014). This finding provides additional support for citrulline consumption conferring antiatherogenic effects by regulating cholesterol homeostasis (Uto-Kondo et al., 2014).

2. S-Allyl Cysteine

S-allyl cysteine is the most abundant bioactive compound from aged garlic extract. S-allyl cysteine exhibits potent antioxidative and anti-inflammatory effects by inhibiting LDL oxidation and NF-κB activation and by counteracting atherogenic events, including foam cell formation (Ho et al., 2001). Furthermore, S-allyl cysteine (10, 20, and 40 mM) can increase the ABCA1 expression in differentiated THP-1 macrophages, indicating a potential to reduce lipid accumulation by promoting cholesterol efflux (Malekpour-Dehkordi et al., 2013).

1. Phellinus Linteus Polysaccharides

Phellinus linteus polysaccharide extracts (PLPEs) have immunomodulatory effects. In lipid-laden THP-1 macrophages, low concentration of PLPEs (from 5 to 20 μg/ml) promoted ApoA-1-mediated cholesterol efflux by upregulating PPARγ/ABCA1(G1) in dose-dependent manner. However, high concentration of PLPEs (up to 100 μg/ml) inhibited ApoA-1-mediated cholesterol efflux, increased the NADPH oxidase-dependent ROS production, and decreased the mitochondrial membrane potential and ATP release (Li et al., 2015d). Thus, the dose is an important consideration for future studies with experimental animals or human patients.

2. Astragalus Polysaccharides

Astragalus polysaccharides (APS) represent the polysaccharide fraction of the medicinal herb Astragalus membranaceus. APS (from 25 to 100 μg/ml) has been shown to promote the ABCA1 expression in foam cells exposed to TNF-α. As a consequence, APS promoted the cholesterol efflux and affected lipid accumulation (Wang et al., 2010d). Further studies indicate that APS (100 μg/ml) reversed TNF-α-induced NF-κB activation in THP-1-derived foam cells. The antiatherosclerotic properties of APS and their molecular mechanisms of action remain to be further elucidated (Wang et al., 2010d).

1. Organosulfur Compounds: Allicin

Allicin is a sulfur-containing compound present in garlic, which has been shown to inhibit cholesterol synthesis in modified liver homogenates (Sendl et al., 1992). It also has inhibitory effects on inducible NO synthase expression in LPS-stimulated mouse macrophages (Dirsch et al., 1998). Moreover, allicin inhibited the LDL oxidation. The lipid-lowering, anti-inflammatory, and antioxidative properties are underlying its atheroprotective effects observed in hyperlipidemic mice and rabbits (Abramovitz et al., 1999; Gonen et al., 2005; Yokoyama et al., 2012). Importantly, allicin (40 mg 3 times daily for 12 weeks) also decreased the carotid intima/media thickness in CAD patients with hyperhomocysteinemia (Liu et al., 2017a). A recent study (Lin et al., 2017) also showed that allicin reduced the TC, FC, and CE levels in THP-1 macrophage-derived foam cells by upregulating the ABCA1-dependent cholesterol efflux via PPARγ/LXRα signaling pathway.

2. Pyranone Derivatives: Asperlin

Asperlin is a natural compound from the marine fungus Aspergillus versicolor LZD4403, which has potent anti-inflammatory activity (Zhou et al., 2017). A recent study showed that asperlin (1–10 μM) has specific protective effects against LPS-induced foam cell formation by promoting cholesterol efflux from RAW264.7 mouse macrophages (Zhou et al., 2017).

3. Anthraquinone Derivatives: Emodin

Emodin is a pharmacologically active compound isolated from the roots of Rheum palmatum (Da Huang). A recent study showed that emodin (5 and 10 μM) promoted ApoA-1-mediated cholesterol efflux from THP-1 macrophages via PPARγ/LXRα/ABCA1 (G1) signaling pathway (Zhou et al., 2008; Fu et al., 2014) and thereby ameliorated diet-induced atherosclerosis in rabbits at the dose of 10 mg/kg body weight (Hei et al., 2006). Emodin was also reported to reduce atherosclerosis in ApoE^−/− mice (Zhou et al., 2008) and rabbits (Hei et al., 2006). In addition, emodin lowered blood glucose, TC, and TG in diabetic and hyperlipidemic rats (Zhao et al., 2009) by inhibiting SREBP-1 and SREBP-2 (Li et al., 2016).

4. Polyacetylene Derivatives: Falcarindiol

Falcarindiol is a common bioactive compound found in some vegetables and medicinal herbs. Falcarindiol (10 μM) has been reported to promote ApoA-1-mediated cholesterol efflux in differentiated macrophages by increasing the mRNA and protein expression of ABCA1 via PPARγ (Wang et al., 2017e). One unique mechanism of falcarindiol is that it not only increased ABCA1 gene expression but also prevented cathepsin-dependent ABCA1 protein degradation (Wang et al., 2017e).

5. Marine Natural Products

In addition to the compounds mentioned above, several marine natural products have also demonstrated an antiatherosclerotic potential by inhibiting foam cell formation. For example, spiromastixones 6 and 14 (10 μM) (Wu et al., 2015a) significantly inhibited oxLDL-induced RAW264.7 foam cell formation in mouse macrophages by inhibiting lipid uptake, as well as promoting cholesterol efflux mediated by HDL and ApoA-1. Mechanistic studies reveal that both compounds significantly reduced the expression of CD36, while upregulating PPARγ/ABCA1 (G1) (Wu et al., 2015a), indicating that both compounds could serve as promising leads with an antiatherogenic potential.

1. Statins

Statins, used as cholesterol-lowering drugs, effectively reduce CVD and mortality in patients with high risk of CVD (Robson, 2008). The mechanism of their action is related to decrease in cholesterol biosynthesis mediated through competitive inhibition of HMG-CoA reductase and their cholesterol-independent pleiotropic effects. Statins are highly efficacious (in lowering atherogenic LDL), safe, and generally well tolerated in patients with CVD. Statins are reported to reduce the risk for developing nonfatal myocardial infarction, ischemic stroke, cardiovascular, and all-cause mortality. Statins have also been reported to promote the stabilization and regression of pre-established atherosclerotic plaques (Toth and Banach, 2019). Clinically used statins can be grouped in two categories, the lipophilic statins (such as simvastatin and atorvastatin) and hydrophilic statins (such as fluvastatin, pravastatin, and rosuvastatin). Although statins share a common mechanism of action by inhibiting HMG-CoA reductase, they differ in terms of chemical structures/water solubility, pharmacokinetic properties (absorption, distribution, metabolism, and excretion), and efficacy of modulating lipids (Schachter, 2005). The lipophilicity/hydrophilicity of statins may have differential effects and mechanisms in suppressing endothelial dysfunction, foam cell formation, and CVD development. For example, a systematic meta-analysis showed that hydrophilic statins, but not hydrophobic statins, significantly reduced the level of asymmetric dimethylarginine (which inhibits NO formation and promotes endothelial dysfunction) (Serban et al., 2015). Another meta-analysis showed that both the hydrophilic and the lipophilic statins showed similar risk reduction for major adverse cardiac events, cardiovascular death, all-cause mortality, and statin-associated muscle symptoms. However, the authors observed that hospitalization caused by CVDs was lower and elevation of alanine aminotransferase was higher in lipophilic statin- than in hydrophilic statin-treated patients (Bytyçi et al., 2017). In a separate study, Kim et al. (2011) observed that treatment of patients with myocardial infarction with lipophilic statins lead to a better short-term cardiovascular outcome; however, both lipophilic and hydrophilic statins lead to a similar reduction of lipids and 1-year cardiovascular outcomes. Therefore, we might expect different clinical outcomes in different patient populations after treatment with lipophilic or hydrophilic statins for different periods.

In cultured macrophages, both lipophilic and hydrophilic statins can ameliorate foam cell formation. For example, rosuvastatin reduces oxLDL-induced foam cell formation in macrophages by reducing CD36 expression, without affecting SR-A expression (Yu et al., 2018). Another study demonstrated that preincubation of THP-1 macrophages with atorvastatin enhanced cholesterol efflux to ApoA-1 and HDL3 dose dependently (Argmann et al., 2005). Atorvastatin also increased PPARγ activity, enhanced LXR activation, and increased ABCA1 expression (Argmann et al., 2005). A recent study showed that reduced protein expression of calpain-1 is accountable for simvastatin-mediated protective effects against foam cell formation (Yang et al., 2016). However, there are several other studies that indicated that simvastatin decreased the ABCA1 protein levels in THP-1 via miRNA-33 (Horie et al., 2010; Niesor et al., 2015). Therefore, overall the effects of statins on macrophage foam cell formation are still controversial. A genome-wide comparison of the differentially expressed genes in macrophage foam cells treated with hydrophilic or lipophilic statins will provide a clear picture of the cardiovascular protective transcriptome of different statins.

2. Nicotinic Acid

Nicotinic acid (NA) has been used for decades as a drug to reduce the progression of atherosclerosis by decreasing LDL-C and increasing HDL-C levels in plasma (Meyers et al., 2004; Carlson, 2005). However, one recent trial suggests that there were no additional clinical effects upon adding niacin to statin therapy during a 36-month follow up period, though HDL-C and TG levels were significantly increased in the patients with atherosclerotic CVDs (Boden et al., 2011). In macrophages from wild-type mice, NA enhanced ABCG1 expression and promoted cholesterol efflux (Lukasova et al., 2011). It is reported that NA increased the production rate of ApoA-1 in both liver and intestinal cells (Rubic et al., 2004; Lamon-Fava et al., 2008), probably through an activation of MAPK and PPAR pathways (Lamon-Fava and Micherone, 2004; Pandey et al., 2008). On the contrary, other studies reported that there are no effects of NA on ApoA-1 production rate (Jin et al., 1997). Furthermore, NA did not exhibit effects on either cholesterol efflux or key RCT gene transcription in THP-1-derived foam cells (Chai et al., 2013).

3. Ezetimibe

Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, has been shown to reduce plasma cholesterol levels and exhibit an antiatherosclerotic effect (Al-Shaer et al., 2004). One study showed that ezetimibe inhibited foam cell formation via the caveolin-1/MAPK signaling pathway, which might be another mechanism of its anti-atherosclerotic effect (Qin et al., 2016).

4. Proprotein Convertase Subtilisin/Kexin Type 9Antibodies

PCSK9 is an enzyme encoded by the PCSK9 gene on chromosome 1 in human beings (Seidah et al., 2003). PCSK9 reduced the amount of LDLRs in hepatocytes by promoting their degradation (Denis et al., 2012). Blocking PCSK9 expression/activity can increase the LDLR amount on the cellular surface and decrease blood LDL-particle concentrations (Weinreich and Frishman, 2014; Joseph and Robinson, 2015). Monoclonal PCSK9 antibodies have been developed as a very effective approach to inhibit PCSK9 and reduce LDL levels (Chaudhary et al., 2017). Several monoclonal antibodies have been or are being tested in clinical studies. Among them, Alirocumab and Evolocumab were recently approved by the Food and Drug Administration for adult patients with heterozygous familial hypercholesterolemia or with clinically significant atherosclerotic CVD requiring additional LDL lowering (Chaudhary et al., 2017). One study displayed that PCSK9 directly decreased cholesterol efflux by the downregulation of ABCA1 expression (Adorni et al., 2017), which might be another mechanism of the anti-CVD effect conferred by inhibition of PCSK9.

1. Glucagon-like Peptide-1 Receptor Agonists

GLP-1 is a gut hormone that activates a G protein-coupled receptor (GLP-1R) in a glucose-dependent manner to stimulate pancreatic β cells and secretion of insulin postprandially (Campbell and Drucker, 2013). GLP-1R agonists (e.g., liraglutide, lixisenatide, albiglutide) or incretin mimetics are used as medicines for the treatment of type 2 diabetes. Recent evidence has suggested that the augmentation of GLP-1R signaling by administration of GLP-1R agonists has beneficial effects on the cardiovascular system in patients with diabetes (Ussher and Drucker, 2012). Further study indicated that GLP-1R signaling induced autophagy, thereby suppressing foam cell formation in nonobese subjects. In obese patients, stimulation of GLP-1R promoted formation of foam cell and production of TNF-α, IL-1β, and IL-6 (Tanaka et al., 2016).

2. High-density Lipoprotein/Apolipoprotein A-1-raising Agents

Current treatment of atherosclerotic CVD is dominated by lowering LDL-C, while increased plasma levels of cholesterol efflux acceptors HDL-C and ApoA-1 expression have been viewed as an alternative therapeutic strategy against CVD for more than three decades (Bailey et al., 2010). The idea to treat CVD by raising HDL comes from a concept relying on the inverse correlation of CVD and human plasma HDL-C (Brewer, 2004), which retrieves excess cholesterol from peripheral cells, including vessel wall macrophages, to the liver for excretion into the bile (Duffy and Rader, 2006). Although there are some animal studies and human trials demonstrating that infusion of HDL or ApoA-1 limit the progression of atherosclerosis (Tall, 1990; Nissen et al., 2003), a randomized, double-blind study involving 15,067 patients at high cardiovascular risk indicated that the CETP inhibitor torcetrapib raised the levels of HDL particles but failed to counteract atherosclerosis (Barter et al., 2007). Two trials have also compared niacin with placebo, added to simvastatin, and although niacin raised HDL-C by ∼15%, there was no clinical benefit to atherosclerosis (Boden et al., 2011; HPS2-THRIVE Collaborative, 2013). These studies indicate the complexity of HDL biology and suggest that not only HDL-C levels but also size, composition, and functionality of the HDL particles may be important. The focus of drug development should shift from raising HDL-C levels to enhancing HDL function. Current knowledge on the role of HDL in preventing various diseases is threefold: 1) HDL itself is not a reliable biomarker/predictor of CVD and/or cancer; 2) the quality/functionality (such as cholesterol efflux assay in patients derived macrophages), rather than the quantity of HDL, is more important for atheroprotection and CVD prevention; and 3) the functionality of HDL plays an important role not only in CVD (such as atherosclerosis), but also in cancer diagnosis/monitoring (Otocka-Kmiecik et al., 2012; Toth et al., 2014; Ganjali et al., 2019; Penson et al., 2019).

RVX-208 (apabetalone) is a selective antagonist of bromodomain and extra terminal (BET) bromodomains (McLure et al., 2013; Picaud et al., 2013). It interferes with the BET protein bromodomain 4 (BRD4), resulting in an increased expression of ApoA-1 in cells, mice, monkeys, and humans (Bailey et al., 2010; Jahagirdar et al., 2014). RVX-208 is being developed as an orally active small molecule for the treatment of CVD (Nicholls et al., 2011). A clinical study showed that RVX-208 increased ApoA-1, pre-β-HDL, and HDL functionality and increased cholesterol efflux in vitro (Bailey et al., 2010). In two Phase II clinical trials, RVX-208 increased the HDL-C and ApoA-1 levels, exhibiting the ability to treat atherosclerosis in patients with established CVD (Johansson et al., 2014). An international, multicenter Phase III trial for treatment of atherosclerosis commenced in October 2015. In 2018, Resverlogix Corp. announced that it has successfully surpassed the planned enrollment target of over 2400 patients in the ongoing Phase III trial (BETonMACE). The scientific community is looking forward to the results of the Phase III trial. The findings discussed here indicate that the regulation of ApoA-1 expression would be a very promising approach for treating atherosclerosis.

One potential therapeutic strategy related to ApoA-1 is the application of small peptides that mimic the ApoA-1 function. ApoA-1 comprises a total of 243 amino acids and involves 10 amphipathic α-helices, which are very important for its interaction with lipids. Peptides that mimic the amphipathic helices in ApoA-1 (ApoA-1 mimetic peptides) are lately used as therapeutic agents (such as 4F, 6F, FX-5A, ATI-5261, ETC-642) (Stoekenbroek et al., 2015). At present, these peptides are being tested in preclinical models, which show that they increase macrophage cholesterol efflux and exhibit antiatherosclerosis action (Stoekenbroek et al., 2015). A small pilot study (ApoA-1 Milano clinical trial) indicated that a complex of recombinant ApoA-1 Milano with phospholipid carriers (ETC-216) significantly reduced percentage and total atheroma volume and plaque thickness in patients (Nissen et al., 2003). Pfizer purchased the producer of ETC-216, Esperion Therapeutics in 2003, in the hope of developing a more effective treatment than ETC-216. However, Pfizer did announce a progress with the development of such product. Currently, no drugs based on ApoA-1 Milano are commercially available.

CSL-111, an rHDL, is composed of human ApoA-1 and soybean phosphatidylcholine, which mimics native HDL (Stoekenbroek et al., 2015). In the “Effect of rHDL on Atherosclerosis-Safety and Efficacy” (ERASE) trial, although CSL-111 remarkably decreased atheroma volume compared with baseline, it induced a high incidence of liver function abnormalities (Tardif et al., 2007). Therefore, further clinical study on CSL-111 was not continued. CSL-112, a second-generation of CSL-111 with no liver toxicity, increased the ApoA-1, pre-β HDL levels, and ABCA1-mediated cholesterol efflux in two Phase I studies (Krause and Remaley, 2013; Easton et al., 2014). In the Phase II trial, infusion of CSL112 for 4 weeks was well tolerated and did not induce any significant side effects in liver or kidney function. CSL112 significantly enhanced cholesterol efflux (Michael Gibson et al., 2016). In 2018, CSL Behring has started the “ApoA-1 Event reducinG in Ischemic Syndromes II” (AEGIS-II) Phase III clinical trial of CSL112, which is a multicenter, double-blind, randomized, placebo-controlled, parallel-group study. The scientific community is looking forward to the results of the Phase III trial of CSL112.

CER-001, a pre-β HDL mimetic, comprises recombinant ApoA-1, diphosphatidylglycerol, and sphingomyelin (Stoekenbroek et al., 2015). In a Phase I trial, infusion of CER-001 was well tolerated and did not show any side effects at any doses (Keyserling et al., 2011). The Phase II trials indicated that CER-001 increased RCT and might significantly decrease aortic atheroma volume. CER-001 has already reached Phase III for the treatment of patients with genetic HDL deficiencies. However, CER-001 has failed in Phase II trial for the treatment of patients with coronary atherosclerosis following acute coronary syndromes (Nicholls et al., 2018). The results show that CER-001 did not produce plaque regression in statin-treated patients following acute coronary syndrome (Nicholls et al., 2018).

3. Bempedoic Acid (ETC-1002)

Bempedoic acid (ETC-1002), a small-molecule inhibitor of ATP citrate lyase, has been shown to reduce the level of low-density lipoprotein (LDL) cholesterol and the development of atherosclerosis in hypercholesterolemic ApoE^−/− mice (Pinkosky et al., 2016), LDLR^−/− mice (Samsoondar et al., 2017), LDLR-deficient miniature pigs (Samsoondar et al., 2017), and statin-intolerant hypercholesterolemic patients (Ballantyne et al., 2018; Laufs et al., 2019). Bempedoic acid, as a prodrug, is converted to the active agent in liver, but not in skeletal muscle; therefore, bempedoic acid may avoid myotoxicity associated with statin therapy (Penson et al., 2017; Ruscica et al., 2019). A recent Phase III clinical trial has shown that treatment with bempedoic acid for 52-weeks added to the maximally tolerated statin therapy significantly lower LDL-C levels without causing higher incidence of overall adverse events (Ray et al., 2019). To date, no information is available whether bempedoic acid can affect foam cell formation. Based on the promising LDL-lowering effects of bempedoic acid, it is plausible that bempedoic acid could also affect lipid uptake and cholesterol efflux.