Background
Coronary artery disease (CAD) is one of the leading causes of mortality and morbidity worldwide. CAD is considered the main cause of death globally because of its high prevalence in developing countries [
1]. Dyslipidemia is one of the most important factors in the pathogenesis of CAD [
2,
3]. Angiopoietin-like proteins (ANGPTLs) are a group of eight proteins that share structural similarity to the members of the angiopoietin protein family. Recent studies showed that genetic and therapeutic antagonism of ANGPTLs in humans and in mice was associated with levels of lipid fractions and atherosclerotic cardiovascular disease [
4,
5].
Angiopoietin-like protein 8 (ANGPTL8), also known as betatrophin [
6], TD26 [
7], re-feeding induced fat and liver [
8], lipasin [
9], and PRO1185 [
10], was identified as a novel hormone, which plays a major role in lipid metabolism [
11]. ANGPTL8 is a new, but atypical member of the ANGPTL family, because it lacks the C-terminal fibrinogen-like domain, but shares a common coiled-coil domain at the N-terminus with ANGPTL3 and ANGPTL4 [
12]. Low frequency missense variants in the ANGPTL4 (E40K) gene protect against the risk of CAD [
13] and ANGPTL3 loss-of-function mutations reduce the risk of CAD in humans [
5]. ANGPTL8 can regulate the cleavage and activity of ANGPTL3. ANGPTL8 coimmunoprecipitates with the N-terminal domain of ANGPTL3 in mouse plasma and increases the appearance of N-terminal ANGPTL3 in cultured hepatocytes [
11].
Patients with diabetes and CAD have remarkably higher levels of ANGPTL8 than those with only diabetes [
14]. However, no studies have investigated the involvement of ANGPTL8 in atherosclerotic disease in Chinese non-diabetic individuals. Therefore, in this study, we investigated circulating full-length ANGPTL8 levels in patients with CAD and the association between ANGPT8 levels and the severity of CAD in Chinese individuals without diabetes.
Discussion
In this study, we found that circulating full-length ANGPTL8 levels in patients with CAD were significantly elevated compared with controls in Chinese non-diabetic individuals. Circulating full-length ANGPTL8 levels were positively associated with the severity of CAD after adjusting for confounding factors. Circulating full-length ANGPTL8 levels were an independent risk factor for CAD.
Dyslipidemia is one of the most important factors in the pathogenesis of CAD, and ANGPTL8 plays an important role in lipid metabolism. ANGPTL8 levels are significantly and positively related to TG and LDL-C levels, but inversely related to HDL-C levels in children and patients with diabetes [
22‐
24]. ANGPTL8 levels are also positively correlated with hepatocellular lipid content [
25] and ANGPTL8 antisense oligonucleotide prevents hepatic steatosis [
26]. A lot of evidence from animal studies also suggests that ANGPTL8 plays a significant role in lipid metabolism [
8,
27]. Previous studies have shown that serum TG levels of ANGPTL8 null mice were one-third of wild-type. Furthermore, knockdown of ANGPTL8 during 3T3-L1 adipogenesis caused approximately a 35% decrease in TG content, but the activity of lipoprotein lipase (LPL), which is an enzyme that hydrolyzes TG circulating in capillaries of adipose tissues and muscle, was increased [
8,
28]. Another study showed that adenoviral ANGPTL8 overexpression in mice increased serum TG levels, while recombinant ANGPTL8 inhibited LPL activity [
29]. ANGPTL3, 4, and 8 show a sequence that binds to LPL, and ANGPTL8 requires ANGPTL3 for its effects on LPL [
30]. Co-expression of ANGPTL3 and ANGPTL8 in mice results in a reduction in circulating ANGPTL3 and an increase in plasma TG levels, whereas plasma TG levels do not change with expression of ANGPTL8 alone [
11]. A previous study demonstrated that ANGPTL3 was specifically correlated with HDL-C, apolipoprotein A-I, and HDL function in female non-diabetic participants [
18]. Inhibition of ANGPTL8 in mice using a monoclonal antibody decreased plasma TG levels and increased LPL activity [
30]. Therefore, ANGPTL8 might play a role in serum lipid metabolism either directly or indirectly (by promoting the cleavage of serum ANGPTL3). However, in our study, there was no association between serum lipids and circulating full-length ANGPTL8 levels. This may be because all of the subjects who were included in the CAD group were taking lipid-lowering drugs (statins or ezetimibe, Additional file
2: Table S1) according to the American College of Cardiology/American Heart Association cardiovascular prevention guidelines. This led to a significant reduction in serum lipid levels in this group.
ANGPTL8 has similar functions to ANGPTL3 and ANGPTL4 because the N-terminal domains of ANGPTL8 share 20% sequence identity with those of ANGPTL3 and ANGPTL4 [
10]. A previous study reported that ANGPTL3 deficiency reduced the risk of coronary heart disease in humans [
5]. A human monoclonal antibody against ANGPTL3 resulted in a greater decrease in atherosclerotic lesion area and necrotic content compared with a control antibody in dyslipidemic mice [
4]. Serum ANGPTL4 and ANGPTL8 levels are increased in patients with hypertension [
31]. ANGPTL4 missense variants (E40K) protect against the risk of CAD [
13]. Genetic knockout of ANGPTL4 protects APOE
−/− mice against development of atherosclerosis and strongly suppresses the ability of the macrophages to become foam cells [
32]. The human monoclonal antibody against ANGPTL3, named evinacumab, has been approved by the Food and Drug Administration as a new drug for treating familial hypercholesterolemia. ANGPTL3 is a new therapeutic target for atherosclerosis [
33] and inhibition of ANGPTL3 can reduce the residual cardiovascular risk [
34]. ANGPTL8 may activate ANGPTL3 [
11]. ANGPTL8 coimmunoprecipitated with the N-terminal domain of ANGPTL3 in mouse plasma, and increased the appearance of N-terminal ANGPTL3 in cultured hepatocytes [
11]. Therefore, ANGPTL8 may be a new therapeutic target for atherosclerotic cardiovascular disease. However, further research is required to determine this possibility.
Serum ANGPTL8 levels are affected by various factors, such as ethnicity and genetic and metabolic status. Lower plasma HDL-C and LDL-C levels are associated with a variant in ANGPTL8 (rs2278426, R59W) in African Americans and Hispanics, but this association is not apparent in European Americans [
11]. In a Chinese Han population, people with the ANGPTL8 rs2278426 (GA/AA) genotype have lower TC and LDL-C levels than those with the GG genotype. However, there were no differences in serum lipid levels identified between the specific genotypes of ANGPTL8 in a Chinese Mulao population [
35]. Fasting inhibits ANGPTL8 expression, and refeeding can highly induce its expression [
11]. Elevated ANGPTL8 levels are associated with cardiometabolic risk factors, such as TC, TG, and LDL-C, but this association is largely dependent on vitamin D status [
36].
Our study showed that circulating ANGPTL8 levels were positively correlated with age (Additional file
3: Table S2), which is consistent with the conclusions reached by Hu et al. [
37] and Abu-Farha et al. [
38]. Epidemiological evidence indicates that blood lipid levels increase with increasing age [
39], which could be the reason for the elevation in ANGPTL8 levels in our study. Our study also showed that there was no association between plasma ANGPTL8 levels and BMI (Additional file
3: Table S2), consistent with a study by Roth et al. [
40]. However, results regarding the association between plasma ANGPTL8 levels and BMI have been inconsistent. Some studies have indicated that ANGPTL8 levels tend to be negatively correlated with BMI [
41] while others observed that plasma ANGPTL8 concentrations positively correlated with BMI [
41]. To avoid a confounding effect, we adjusted for age and BMI in this study. It is reported that circulating ANGPTL8 levels is increased in patients with polycystic ovary syndrome, partly because of the levels of sex hormone [
42]. In this study, we found that circulating ANGPTL8 levels is higher in men compared with women in the control group (492.73 ± 148.42 vs 361.38 ± 118.44 pg/ml, P = 0.002), while there were no differences in the levels of circulating ANGPTL8 between the two genders in patients with CAD (667.51 ± 238.24 vs 658.93 ± 273.90 pg/ml, P = 0.912). In order to avoid a confounding effect, we also adjusted for sex in this study. A previous study showed that serum ALT levels are positively associated with ANGPTL8 levels [
43], while serum ALT levels were higher in CAD compared with controls in our study (Table
1). Therefore, ALT was adjusted in this study. We also found that serum creatinine was positively associated with ANGPTL8 in this study (Additional file
3: Table S2). Renal function is independently associated with circulating ANGPTL8 and circulating ANGPTL8 levels are positively correlated with serum creatinine levels [
44]. However, the mechanism for the association between serum creatinine and ANGPTL8 levels is unknown. We adjusted for serum creatinine levels in this study.
Care was taken to avoid bias in this study. An enzyme-linked immunosorbent assay was performed according to the manufacturer’s instructions by a trained experimenter who was unaware of patients’ clinical data. Moreover, in statistical analysis, adjustments were made for the confounding effects of risk factors for CAD and circulating ANGPTL8 levels. Finally, propensity score matching was used to reduce the effects of outcome-selection bias.
This study has some limitations. First, it was a case–control study, which meant that it could only show associations, not causality. Second, all of the patients with CAD and some controls were taking drugs (Additional file
2: Table S1). The effects of medication on ANGPTL8 levels were not observed in this study. Third, because all of the study participants were Chinese, the findings may not be generalizable to other ethnicities. Our findings should be confirmed in other populations.