APOA1/C3/A4/A5 Gene Cluster at 11q23.3 and Lipid Metabolism Disorders: From Epigenetic Mechanisms to Clinical Practices

The APOA1/C3/A4/A5 cluster is an essential component in regulating lipoprotein metabolism and maintaining plasma lipid homeostasis. A genome-wide association analysis and Mendelian randomization have revealed potential associations between genetic variants within this cluster and lipid metabolism disorders, including hyperlipidemia and cardiovascular events. An enhanced understanding of the complexity of gene regulation has led to growing recognition regarding the role of epigenetic variation in modulating APOA1/C3/A4/A5 gene expression. Intensive research into the epigenetic regulatory patterns of the APOA1/C3/A4/A5 cluster will help increase our understanding of the pathogenesis of lipid metabolism disorders and facilitate the development of new therapeutic approaches. This review discusses the biology of how the APOA1/C3/A4/A5 cluster affects circulating lipoproteins and the current progress in the epigenetic regulation of the APOA1/C3/A4/A5 cluster.


Introduction
Dyslipidemia is a commonly encountered chronic condition in clinical practice involving abnormalities in plasma cholesterol, triglycerides (TGs), or both [1].Dyslipidemia is also an important risk factor for atherosclerotic cardiovascular diseases (ASCVDs), such as coronary artery disease (CAD), stroke, and peripheral vascular disease [2].Elevated levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), TGs, and lipoprotein A, along with decreased high-density lipoprotein cholesterol (HDL-C), are major contributors to ASCVD risk [3].Systemic metabolic diseases, including obesity and diabetes, are intimately associated with dyslipidemia [4,5].Dyslipidemia leads to the intrahepatic accumulation of fat, resulting in non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and liver fibrosis [6,7].Additionally, severe hypertriglyceridemia (HTG) results in critical clinical conditions, including acute pancreatitis and familial chylomicronemia syndrome (FCS) [8,9].Therefore, in the primary and secondary prevention of lipid disorders, the treatment of dyslipidemia plays an important role [10].
Recently, SNPs in APOA1 have been widely utilized as predictive markers for CAD risk, and prospective studies of weight loss in obese patients have shown that the APOA1 (rs670) gene displays significant effects on LDL cholesterol levels and insulin resistance [14][15][16].Largescale population genetic studies have shown that loss-of-function (LOF) mutations in the Biomedicines 2024, 12, 1224 2 of 14 APOC3 gene confer reduced very-low-density lipoprotein (VLDL) and non-HDL cholesterol levels, which are associated with a decreased risk of HTG and coronary atherosclerosis [17,18].APOA4 gene polymorphisms serve as important indicators for renal and cerebral vascular diseases [19,20].Several meta-analyses have shown that SNPs in APOA5 may be important contributors to the elevated risk of cardiovascular disease.Specifically, compared with carriers of the T allele, carriers of the APOA5 rs662799-C allele have higher plasma TC and TG levels and lower plasma HDL-C levels [21,22].Neighboring genes of the APOA1/C3/A4/A5 gene cluster, including BUD13, ZPR1, and SIK1, have also been implicated in lipid metabolism [23,24].SNPs in the BUD13 and ZPR1 genes have strong associations with elevated triglyceride levels in patients with metabolic syndrome [25].SIK1 is expressed in liver tissue and may affect lipid metabolism by regulating fatty acid synthesis and catabolism pathways [26].In conclusion, the APOA1/C3/A4/A5 cluster holds potential as a novel target for addressing lipid metabolism disorders (Figure 1).Recently, SNPs in APOA1 have been widely utilized as predictive markers for CAD risk, and prospective studies of weight loss in obese patients have shown that the APOA1 (rs670) gene displays significant effects on LDL cholesterol levels and insulin resistance [14][15][16].Large-scale population genetic studies have shown that loss-of-function (LOF) mutations in the APOC3 gene confer reduced very-low-density lipoprotein (VLDL) and non-HDL cholesterol levels, which are associated with a decreased risk of HTG and coronary atherosclerosis [17,18].APOA4 gene polymorphisms serve as important indicators for renal and cerebral vascular diseases [19,20].Several meta-analyses have shown that SNPs in APOA5 may be important contributors to the elevated risk of cardiovascular disease.Specifically, compared with carriers of the T allele, carriers of the APOA5 rs662799-C allele have higher plasma TC and TG levels and lower plasma HDL-C levels [21,22].Neighboring genes of the APOA1/C3/A4/A5 gene cluster, including BUD13, ZPR1, and SIK1, have also been implicated in lipid metabolism [23,24].SNPs in the BUD13 and ZPR1 genes have strong associations with elevated triglyceride levels in patients with metabolic syndrome [25].SIK1 is expressed in liver tissue and may affect lipid metabolism by regulating fatty acid synthesis and catabolism pathways [26].In conclusion, the APOA1/C3/A4/A5 cluster holds potential as a novel target for addressing lipid metabolism disorders (Figure 1).APOE, APOC3, and so on to assemble into VLDL.CM and VLDL are synthesized and transported into the blood and circulated, where they are hydrolyzed by LPL to generate fatty acids, VLDL remnants, and CM remnants.The fatty acids are internalized and used as an energy source in extrahepatic organs or for the storage of energy in adipocytes.Part of the CM remnants is ingested by the APOE receptor (APOER) and broken down in the liver, while part of the VLDL remnants is further converted to LDL.ABCA1, located on the surface of the peripheral cell membrane, transports excess free cholesterol and triglyceride into the circulating blood, which then combines with APOA1 to form nascent high-density lipoprotein (nHDL).Subsequently, nHDL is converted to spherical mature high-density lipoprotein (mature HDL) by esterification with LCAT.Mature HDL can be removed by SR-BI, or it can further transfer cholesterol to LDL by CETP.LDL produced by endogenous and exogenous pathways can be recognized and taken up by LDLR and then enter the liver for catabolism.APOC3 on the surface of LDL can displace APOE or directly interfere with the binding of APOE to LDLR, thereby inhibiting the hepatic clearance of LDL.The abnormal accumulation of CM and VLDL remnants in the subendothelium of the impaired arteries induces the production of various cytokines, inflammatory mediators, and biological enzymes, which promote the differentiation of macrophages.Differentiated macrophages phagocytize lipid particles and become foam cells, which die to form lipid pools and eventually develop into atherosclerotic plaques.
The heredity of most organisms is based on the inheritance of genomic DNA [27].Although the relationship between the genetic variability and lipid metabolism of APOA1/C3/A4/A5 has been extensively elucidated, there remain functional changes and disease consequences that cannot be solely accounted for by gene mutations [11,12].Notably, numerous studies have identified the importance of epigenetic processes in regulating APOA1/C3/A4/A5 gene expression (Figure 2).Epigenetic modifications are heritable changes in gene expression caused by environmental factors without altering the DNA sequence.Epigenetic modifications mainly include DNA methylation, histone methylation, histone acetylation, RNA modification, and non-coding RNA [28].This epigenetic control is critical to the physiological control of transcription and translation processes [27].As epigenetic controls of APOA1/C3/A4/A5 have been continually reported in recent years, numerous novel insights into diagnosis, treatment, and prognosis originate from epigenetics.Therefore, this paper comprehensively investigates the diverse mechanisms underlying epigenetic regulation in the APOA1/C3/A4/A5 gene cluster and discusses emerging strategies for treating lipid metabolism disorders.
of LDL can displace APOE or directly interfere with the binding of APOE to LDLR, thereby inhibiting the hepatic clearance of LDL.The abnormal accumulation of CM and VLDL remnants in the subendothelium of the impaired arteries induces the production of various cytokines, inflammatory mediators, and biological enzymes, which promote the differentiation of macrophages.Differentiated macrophages phagocytize lipid particles and become foam cells, which die to form lipid pools and eventually develop into atherosclerotic plaques.
The heredity of most organisms is based on the inheritance of genomic DNA [27].Although the relationship between the genetic variability and lipid metabolism of APOA1/C3/A4/A5 has been extensively elucidated, there remain functional changes and disease consequences that cannot be solely accounted for by gene mutations [11,12].Notably, numerous studies have identified the importance of epigenetic processes in regulating APOA1/C3/A4/A5 gene expression (Figure 2).Epigenetic modifications are heritable changes in gene expression caused by environmental factors without altering the DNA sequence.Epigenetic modifications mainly include DNA methylation, histone methylation, histone acetylation, RNA modification, and non-coding RNA [28].This epigenetic control is critical to the physiological control of transcription and translation processes [27].As epigenetic controls of APOA1/C3/A4/A5 have been continually reported in recent years, numerous novel insights into diagnosis, treatment, and prognosis originate from epigenetics.Therefore, this paper comprehensively investigates the diverse mechanisms underlying epigenetic regulation in the APOA1/C3/A4/A5 gene cluster and discusses emerging strategies for treating lipid metabolism disorders.

Function of APOA1
APOA1 is the major protein fraction of high-density lipoprotein (HDL) particles, playing a pivotal role in HDL synthesis [29] (Figure 1).HDL, commonly regarded as "good cholesterol", exerts beneficial effects such as reducing cardiovascular risk by

APOA1: Proteins with Therapeutic Potential for Lipid Metabolism Disorders 2.1. Function of APOA1
APOA1 is the major protein fraction of high-density lipoprotein (HDL) particles, playing a pivotal role in HDL synthesis [29] (Figure 1).HDL, commonly regarded as "good cholesterol", exerts beneficial effects such as reducing cardiovascular risk by facilitating reverse cholesterol transport (RCT) [30,31].HDL can help remove excess cholesterol from peripheral tissues to the liver, where it undergoes clearance and subsequent excretion via bile [32].In addition, HDL acts as an inhibitor of atherosclerotic, inflammatory, and apoptotic properties, which, together, enhance its protective role against ASCVD [33,34].
APOA1 is primarily produced by hepatocytes and then released into the bloodstream [29].APOA1 interacts with the ATP-binding cassette transporter A1 (ABCA1) and triggers cholesterol efflux, promoting the transfer of intracellular cholesterol and phospholipid to nascent high-density lipoprotein (nHDL) [35][36][37].Subsequently, APOA1 interacts with lecithin-cholesterol acyltransferase (LCAT) to activate LCAT and promote the conversion of nHDL to mature HDL [38].Mature HDL can be selectively removed from plasma by the hepatic HDL receptor, scavenger receptor type BI (SR-BI).Cholesterol in HDL can also be transferred to TRLs, including very-low-density lipoprotein (VLDL) and LDL, under the action of cholesteryl ester transfer proteins (CETPs), and then it can be transported to the liver by low-density lipoprotein receptors (LDLRs) [36,37,39].
During the progression of atherosclerotic disease, vascular endothelial cells release proinflammatory signals, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (vCAM-1), which significantly contribute to the initiation of early lesions.These molecules are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs) and mediate leukocyte adherence to endothelial cells during atherosclerosis and myocardial infarction [40].Recent studies have demonstrated that the injection of APOA1 into rabbits can downregulate the expression levels of VCAM-1 and ICAM-1, thus exerting anti-inflammatory and anti-atherosclerotic effects [41].APOA1 also has potent anti-inflammatory effects on macrophages through the rapid disruption of lipid rafts and the inhibition of the PI3K/AKT pathway, which significantly reduces macrophage chemotaxis [42].Simultaneously, facilitated by the APOA1 binding protein (AIBP), APOA1 tightly binds to ABCA1 on the macrophage membrane, stabilizing the ABCA1 protein against COP9 signalosome subunit 2 (CSN2)-mediated degradation.This process prevents foam cell formation [43].Together, these findings suggest that APOA1 has the ability to attenuate inflammatory cytokine production in endothelial cells and macrophages, thereby exhibiting anti-inflammatory properties.

Epigenetic Regulation and Therapeutic Potential of APOA1
The regulation of APOA1 is complex and occurs at different stages of gene expression (Figure 2).APOA1-AS, the natural antisense strand of APOA1, induces the silencing of APOA1 gene expression by recruiting lysine (K)-specific demethylase 1 (LSD1) to catalyze the demethylation of tri-methylated histone H3 lysine 4 (H3K4) in the promoter region of APOA1.However, the antisense oligonucleotide (ASO) targeting APOA1-AS leads to the upregulation of APOA1 expression both in vitro and in vivo, suggesting that APOA1-AS is an efficient transcriptional regulator of APOA1 [44].Given its high specificity, stability, and efficiency, ASO targeting APOA1-AS may emerge as an effective lipid-lowering drug in the near future [45].
APOA1-AS is also crucial for regulating coronary atherosclerosis.In vascular smooth muscle cells (VSMCs) treated with oxidized low-density lipoprotein (ox-LDL), the expression of APOA1-AS is significantly upregulated.Conversely, the absence of APOA1-AS impairs the proliferation and migration of ox-LDL-VSMCs and stimulates cell apoptosis.Mechanistically, APOA1-AS recruits TATA-box binding protein-related factor 15 (TAF15) to stabilize SMAD family member 3 (SMAD3) mRNA, subsequently activating the TGFβ/SMAD3 signaling pathway to promote VSMC proliferation and migration.In addition, APOA1-AS inhibits VSMC apoptosis, highlighting its therapeutic potential for coronary atherosclerosis [46][47][48].
Cholesterol participates in reproductive processes by being a precursor for the synthesis of synthetic sex hormones.Disorders in lipid metabolism within sexual organs can result in fluctuations in sex hormone levels, subsequently impacting the growth or functionality of reproductive organs [49].Bisphenol A, a widely utilized plasticizer found abundantly in aquatic environments, exerts numerous adverse effects on reproduction.The latest research has demonstrated that bisphenol A can induce the hypermethylation of CpG sites in the promoter region of APOA1, significantly elevating the transcription level of APOA1 within the testes and enhancing testicular reverse cholesterol transport.Prolonged exposure to bisphenol A further increases HDL-C levels within the testes while significantly inhibiting TC and free cholesterol levels, leading to disruptions in sex hormone balance and the subsequent impairment of fish spermatogenesis [50].

Function of APOC3
APOC3, which is primarily expressed in hepatocytes, is an apolipoprotein of 79 amino acid residues and is an important component of VLDL and HDL [51].APOC3 has long been considered one of the most critical factors in TRL metabolism, which is closely related to the increase in the plasma TG level (Figure 1).As a co-factor of LPL, APOC3 is essential for regulating TRL lipolysis.The lipolysis efficiency of the LPL enzyme is related to the ratio of APOC2 and APOC3 on the particle surface [52].APOC3 is a competitive inhibitor of the APOC2 activation of LPL, inhibiting LPL enzyme activity and preventing the degradation of TRLs [53].APOC3 also blocks the binding of the LPL enzyme to glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1) on vascular endothelial cells.This prevents the anchoring of the LPL enzyme on the vascular endothelium, further amplifying APOC3's inhibitory effect on triglyceride lipolysis [54].In the liver, APOC3 facilitates VLDL assembly and secretion.The hepatic assembly of VLDL is achieved by recruiting large amounts of triglycerides as lipid droplets in the microsomal lumen to the VLDL precursors containing APOB-100.APOC3 promotes the binding of triglycerides to APOB-100 and the secretion of mature VLDL [55].In addition, APOC3 displaces APOE from TRL or directly inhibits the binding of APOE to LDLR and LDLR-related protein 1 (LRP1) on the liver surface, thereby inhibiting the hepatic clearance of TRLs [56].Notably, the CRISPR/Cas9 system was used to construct APOC3-KO rabbits, and knocking down APOC3 in these rabbits led to a 50% decrease in the TG level with a significant increase in LPL activity.Moreover, APOC3 deficiency was found to be more beneficial in maintaining the TC, TG, and LDL-C levels, inhibiting inflammatory responses, and preventing atherosclerosis when fed a high-fat diet [57].

Epigenetic Regulation and Therapeutic Potential of APOC3
APOC3 is the most important regulator in triglyceride metabolism, which further affects glucose and lipid metabolism, atherosclerotic plaque formation, inflammation, and endothelial endoplasmic reticulum stress response.APOC3 is an important factor in the development of lipodystrophy, diabetic dyslipidemia, and coronary artery calcification [58][59][60][61].Epidemiological studies and observational research have shown that increased levels of APOC3 lead to increased TG levels and a high risk of ASCVD, while LOF mutations in APOC3 are linked to decreased plasma TG levels and reduced cardiovascular disease risk among high-risk individuals [62,63].For example, rs4225 in the 3'UTR region of the APOC3 gene is the small ribonucleic acid binding site.The T allele of rs4225, but not the G allele, inhibited APOC3 expression by promoting the binding of miR-4271 to the 3'UTR of APOC3 mRNA.Individuals with the GG genotype exhibited elevated plasma APOC3 levels compared to individuals possessing the TT genotype.Patients with the G allele have a higher multivessel incidence and a greater incidence of left anterior descending artery disease, thus showing more severe ASCVD, which provides evidence that miR-4271 regulates APOC3 expression levels and lipid metabolism [64] (Table 1).
Given that APOC3 is an important target for reductions in TG and ASCVD risk, RNA interference therapies such as ASO and small interfering RNA (siRNA) techniques have been used to target APOC3 mRNA [70].ASO binds to the target mRNA and hydrolyzes the RNA strand by activating RNase.Different from ASO, double-stranded siRNA separates into two strands in the cytoplasm, with one strand hybridizing and degrading mRNA.N-acetyl galactosamine (GalNAc) is a carbohydrate with a high affinity to receptors on hepatocytes.Current studies often combine ASO or siRNA with GalNAc to improve the specific uptake of the liver so as to improve the efficacy under the condition of an equal dose and limit the off-target side effects [71].ARO-APOC3, an siRNA drug targeting APOC3, has been proposed for the treatment of FCS.Phase 3 clinical trial results show that after 16 weeks of intervention, ARO-APOC3 treatment reduced LDL-C by 53% and TG by 55% without serious adverse events [72,73].Volanesorsen, an anti-APOC3 ASO drug, has also entered the clinical trial stage.The results from phase 3 clinical trials demonstrate that Volanesorsen exhibits significant efficacy in reducing TG levels and decreasing the occurrence of acute pancreatitis events among patients with HTG.Consequently, it has received approval for treating FCS [74,75].Another novel GalNAc-conjugated ASO drug, Olezarsen, has not only been proven to reduce TG levels in individuals with mild TG elevation but has also demonstrated good efficacy in patients with TG levels ranging from 200 to 500 mg/dL and a high risk of ASCVD.It has been shown to significantly reduce the risk of cardiovascular disease in patients with moderate to severe HTG with excellent safety and tolerability [71,76].In extravascular lipid metabolism disorders, targeting APOC3 therapy also has significant efficacy.An assessment of the hepatic fat fraction (HFF) in patients with severe HTG, familial partial lipodystrophy, and FCS after 12 months of Volanesorsen treatment showed that, compared with the controls, the HFF of the treatment group was significantly reduced by 3.02-8.34%.These results indicate that inhibiting hepatic APOC3 synthesis has a good therapeutic effect on HFF [77].
Recently, numerous studies have reported epigenetic modifications of DNA and RNA targeting APOC3.DNA methylation marks of key genes are closely linked to inflammatory cell drive, apoptosis, thrombosis, and atherogenic signaling, as a recent study in a Chinese population with acute coronary syndrome has shown [78].The identification of APOC3 promoter methylation levels in patients with CAD showed that the CpG islands in the promoter region were hypermethylated [79].In addition, METTL3 enhanced APOC3 mRNA stability, increased the APOC3 transcription level, and induced APOC3 protein expression in an m 6 A-dependent manner, while si-METTL3 reduced APOC3 mRNA stability and then inhibited APOC3 protein expression [80].Taken together, these studies provide evidence of the DNA methylation status and m 6 A modification of the APOC3 gene (Figure 2).

Function of APOA4
APOA4 is a plasma lipoprotein that is primarily synthesized by the liver, as well as the small intestine, and subsequently secreted into the bloodstream (Figure 1).Plasma APOA4 is mainly distributed on the surface of chylomicrons and HDL, or it exists in a lipoprotein-free form.APOA4 is composed of 12 amphipathic helices that mediate lipid binding and interactions with aqueous humor.These helices are arranged to create a central hydrophobic pocket that can accommodate lipids, making them both adsorptive and exchangeable [81].
APOA4 is involved in lipid metabolism and glucose metabolism.As an exchangeable apolipoprotein, APOA4 participates in the secretion and clearance of chylomicrons and regulates the absorption of dietary fat [82].Common APOA4 mutations include N147S, T347S, and Q360H [83].A study by Hockey et al. showed that the 360H allele has greater lipid affinity compared with 360Q [84].APOA4 also has an anti-atherosclerotic effect.In APOE −/− mice, a plasma lipid measurement and a quantitative analysis of aortic lesions indicated that the high expression of APOA4 in the intestine can reduce blood lipids and inhibit oxidative damage in local arterial tissues, thereby delaying the progression of atherosclerosis [85][86][87].APOA4 also ameliorates hyperglycemia by increasing insulin secretion and glucose uptake activation in adipocytes.Further studies showed that APOA4 inhibited hepatic steatosis by down-regulating SREBF1-mediated lipogenesis and improved hepatic insulin sensitivity through IRS-PI3K-Akt signaling, thus ameliorating NAFLD [88].In addition, APOA4 also plays an important anti-inflammatory role.Related studies have shown that APOA4 powerfully inhibits ROS activity.Moreover, the inhibition of APOA4 can suppress the activity of immune cells and reduce immune cell infiltration, further indicating that APOA4 has a bi-functional effect in regulating inflammatory injury and immune cell infiltration [89].The overexpression of APOA4 attenuated liver injury by inhibiting the secretion of liver fibrosis mediators and inflammatory factors in CCL4induced liver injury in mice, controlling the levels of antioxidant enzymes, and reducing the proportion of pro-inflammatory monocytes [90,91].Thus, APOA4 may be a potential new therapeutic target for treating liver injury.

The Epigenetic Regulation and Therapeutic Potential of APOA4
Concerning the epigenetic regulation of APOA4, the lncRNA APOA4-AS has been identified as a co-regulator of APOA4 expression.APOA4 genes and APOA4-AS have similar expression patterns.The expressions of both APOA4-AS and APOA4 were found to be abnormally elevated in the livers of ob/ob mice and in patients with fatty liver disease.The knockout of APOA4-AS reduced APOA4 expression and resulted in lower plasma triglyceride and cholesterol levels in ob/ob mice.Mechanistically, APOA4-AS directly interacts with HUR to stabilize APOA4 mRNA, whereas HUR loss significantly reduces APOA4 transcripts [92].Another nuclear lncRNA, Lnc19959.2,binds specifically to the APOA4 transcriptional repressor Purb and promotes Purb ubiquitination and degradation, leading to increased APOA4 expression [93].
APOA4, as a biomarker, plays an important role in disease prediction.Stephens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are rare but severe adverse drug reactions.A plasma proteomic analysis showed that APOA4 expressed significantly differentially between the treatment and control groups, and a multivariate regression analysis showed that the APOA4 levels were highly associated with the prognostic parameters of SJS/TEN.Therefore, APOA4 can be used as a prognostic marker for SJS/TEN [94].Of note, APOA4 also serves as a valuable predictor of the residual risk for coronary heart disease (CHD) and guides emerging preventive therapies against CHD.In the PROCARDIS CHD case-control study, the association between apolipoproteins and CHD risk was quantified using a single mass spectrometry assay.An elevated APOA4 level was inversely associated with CHD risk, which was confirmed to be an associated factor for CHD independent of HDL.The detection and identification of APOA4 will help to lower the residual risk in patients with CHD [95].
The pathogenesis of NAFLD is complex.Epigenetic modifications, especially DNA methylation changes, have been intensively studied in recent years [96,97].APOA4 exhibited DNA hypomethylation and had significantly higher expression in the liver of Biomedicines 2024, 12, 1224 8 of 14 HFD-treated mice.This suggests that the assessment of the APOA4 DNA methylation status and gene expression can be used to diagnose NAFLD and its severity [98] (Figure 2).

Function of APOA5
APOA5 has been recognized as one of the most potent factors affecting plasma TGs [99].APOA5, synthesized by the liver, is important for the maturation and secretion of VLDL and facilitates the formation of hepatic lipid droplets (LDs) [100,101].Notably, APOA5 exerts an important function of lowering plasma triglycerides despite its low concentration (Figure 1).
Hepatic APOA5 can use fatty acids derived from adipocytes to promote the synthesis of LDs, thereby promoting lipid deposition in hepatocytes [102].A series of clinical and in vivo experiments have proven that APOA5 can play a positive role in NALFD, and knocking down the intracellular expression of APOA5 leads to a significant reduction in intracellular TG.In the development of insulin resistance and obesity, APOA5 may act as a sensor for fatty acid accumulation in adipocytes, resulting in an increased number and increased size of LDs in the liver [103,104].Thus, APOA5 may serve as an important regulator of TG storage in hepatocytes [105,106].
In plasma, APOA5 on the surface of TRLs enhances triglyceride hydrolysis and cholesterol remnant clearance.On one hand, APOA5 interacts with the ANGPTL3/8 complex and selectively blocks the inhibitory effect of the ANGPTL3/8 complex on LPL in a concentration-dependent manner [107].On the other hand, APOA5 can also interact with glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) to regulate LPL activity.GPIHBP1 is an endothelial membrane protein that facilitates the transfer of LPL from LPL-producing cells, including myocytes and adipocytes, to the vascular surface of the capillary endothelium, allowing LPL to function on the vascular surface.APOA5 interacts with GPIHBP1 to help TRLs adhere to the endothelial cell surface, and it promotes LPL-mediated TG hydrolysis [108].Moreover, circulating APOA5 can also bind to the LDLR family and heparan sulfate proteoglycan (HSPG) family to effectively promote the removal of cholesterol remnants from circulation [109,110].In conclusion, APOA5 maintains lipid homeostasis through multiple dimensions, and it especially plays a key role in TG homeostasis [100].

Epigenetic Regulation and Therapeutic Potential of APOA5
Many epigenetic factors, especially miRNA, regulate the expression of APOA5.It has been reported that the C allele of rs2266788 of the APOA5 gene can generate a potential microRNA-binding site in the 3'UTR of APOA5, which is specifically recognized by liverexpressed miR-485-5p, leading to the down-regulation of the hepatic transcription of APOA5 and the increase in the plasma TG level.Similarly, miR-3201 specifically bound to the T allele of rs2266788 and negatively regulated the transcriptional activity of APOA5.Reversely, the inhibition of miR-3201 expression significantly increased APOA5 expression in HepG2 cells [68] (Table 1).
As a key molecule in triglyceride metabolism, the epigenetic heterogeneity of APOA5 partially explains individual susceptibility to HTG.In patients with HTG, the APOA5 promoter and exon 3 were hypermethylated.There was a significant positive correlation between exon 3 methylation and TG levels, and it is also positively associated with atherosclerotic dyslipidemia.Furthermore, the methylation rate of exon 3 exhibited a remarkable prevalence of 82% in patients with HTG harboring APOA5 SNPs.Collectively, exon 3 CGI methylation in APOA5 acts synergistically with genetic polymorphisms to increase the risk of HTG [111].
Recently, the prevalence of childhood obesity has been increasing, which has become a public health issue.Methylation is a key regulator of gene-environment interactions, and it is closely related to obesity.A genome-wide methylation array analysis of patients with obesity and controls revealed a significant inverse association between the level of methylation within the APOA5 locus and obesity.It can be concluded that altered methylation at the CpG sites of specific genes, especially the altered methylation of genes regulating lipoprotein expression, may lead to childhood obesity.It also provides a new understanding of the etiology of obesity [112] (Figure 2).

Conclusions
Dyslipidemia, defined as high levels of lipids (TC, TG, or both) or low levels of HDL-C, directly increases the risk of many diseases including atherosclerosis, fatty liver disease, and acute pancreatitis.Among them, ASCVD deserves our attention.The effective treatment and management of this patient population has been a major challenge for the global medical community for many years due to the potential risk of high-risk cardiovascular events.Despite the great achievements of LDL-C-lowering drugs in reducing the risk of ASCVD, dyslipidemia characterized by elevated TRL levels still contributes to substantial residual cardiovascular risk and is increasing worldwide due to obesity and aging [17].Of note, addressing the residual cardiovascular risk caused by high TRLs is a new direction for lipid management in the future.
Since the beginning of the 21st century, the development of gene therapy technologies, from traditional gene replacement to gene editing, has opened up endless possibilities for the treatment of dyslipidemia diseases.Besides gene editing systems such as CRISPR/Cas9, emerging epigenetic modulation techniques such as ASO and RNAi therapy are showing promise in regulating lipid homeostasis.The level of epigenetic modification of related genes can also play a role in the prediction and early warning of lipid metabolism disorders.Moreover, most phenotypically relevant sites were identified and translated into diagnostic assays for routine use in clinical utility along with advanced mapping methods for DNA and RNA modification.APOA1/C3/A4/A5 plays an important role in lipid metabolism.Recent insights into APOA1/C3/A4/A5 epigenetic mechanisms have increased our understanding of lipid metabolism.These findings provide new ideas for the prevention of lipid disorders and the reduction in residual cardiovascular risk, provide more effective and low-risk treatment methods, and fundamentally reduce the global economic burden associated with lipid metabolism disorders.

Figure 1 .Figure 1 .
Figure 1.Dietary triglycerides and cholesterol are absorbed in the gastrointestinal tract and enter small intestinal epithelial cells.Cholesterol and triglycerides are assembled into CM through a synthetic pathway dependent on a microsomal triglyceride transporter and APOB-48 with the assistance of APOE, APOC3, APOA4, and APOA5.Endogenous triglyceride and cholesterol are synthesized in the liver and then closely combined with APOB-100, APOE, APOC3, and so on to assemble into VLDL.CM and VLDL are synthesized and transported into the blood and circulated, where they are hydrolyzed by LPL to generate fatty acids, VLDL remnants, and CM remnants.The fatty acids are internalized and used as an energy source in extrahepatic organs or for the storage of energy in adipocytes.Part of the CM remnants is ingested by the APOE receptor (APOER) and broken down in the liver, while part of the VLDL remnants is further converted to LDL.ABCA1, located on the surface of the peripheral cell membrane, transports excess free cholesterol and triglyceride into the circulating blood, which then combines with APOA1 to form nascent high-density Figure 1.Dietary triglycerides and cholesterol are absorbed in the gastrointestinal tract and enter small intestinal epithelial cells.Cholesterol and triglycerides are assembled into CM through a synthetic pathway dependent on a microsomal triglyceride transporter and APOB-48 with the assistance of APOE, APOC3, APOA4, and APOA5.Endogenous triglyceride and cholesterol are synthesized in the liver and then closely combined with APOB-100,APOE, APOC3, and so on to assemble into VLDL.CM and VLDL are synthesized and transported into the blood and circulated, where they are hydrolyzed by LPL to generate fatty acids, VLDL remnants, and CM remnants.The fatty acids are internalized and used as an energy source in extrahepatic organs or for the storage of energy in adipocytes.Part of the CM remnants is ingested by the APOE receptor (APOER) and broken down in the liver, while part of the VLDL remnants is further converted to LDL.ABCA1, located on the surface of the peripheral cell membrane, transports excess free cholesterol and triglyceride into the circulating blood, which then combines with APOA1 to form nascent high-density lipoprotein (nHDL).Subsequently, nHDL is converted to spherical mature high-density lipoprotein (mature HDL) by esterification with LCAT.Mature HDL can be removed by SR-BI, or it can further transfer cholesterol to LDL by CETP.LDL produced by endogenous and exogenous pathways can be recognized and taken up by LDLR and then enter the liver for catabolism.APOC3 on the surface of LDL can displace APOE or directly interfere with the binding of APOE to LDLR, thereby inhibiting the hepatic clearance of LDL.The abnormal accumulation of CM and VLDL remnants in the subendothelium of the impaired arteries induces the production of various cytokines, inflammatory mediators, and biological enzymes, which promote the differentiation of macrophages.Differentiated macrophages phagocytize lipid particles and become foam cells, which die to form lipid pools and eventually develop into atherosclerotic plaques.