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Myocardial infarction (MI) triggers a potent inflammatory response via the release of circulatory mediators, including extracellular vesicles (EVs) by damaged cardiac cells, necessary for myocardial healing. Timely repression of inflammatory response are critical to prevent and minimize cardiac tissue injuries, nonetheless, progression in this aspect remains challenging. The ability of EVs to trigger a functional response upon delivery of carried bioactive cargos, have made them clinically attractive diagnostic biomarkers and vectors for therapeutic interventions. Using label-free quantitative proteomics approach, we compared the protein cargo of plasma EVs between patients with MI and from patients with stable angina (NMI). We report, for the first time, the proteomics profiling on 252 EV proteins that were modulated with >1.2-fold after MI. We identified six up-regulated biomarkers with potential for clinical applications; these reflected post-infarct pathways of complement activation (Complement C1q subcomponent subunit A (C1QA), 3.23-fold change, p = 0.012; Complement C5 (C5), 1.27-fold change, p = 0.087), lipoprotein metabolism (Apoliporotein D (APOD), 1.86-fold change, p = 0.033; Apolipoprotein C-III (APOCC3), 2.63-fold change, p = 0.029) and platelet activation (Platelet glycoprotein Ib alpha chain (GP1BA), 9.18-fold change, p < 0.0001; Platelet basic protein (PPBP), 4.72-fold change, p = 0.027). The data have been deposited to the ProteomeXchange with identifier PXD002950. This novel biomarker panel was validated in 43 patients using antibody-based assays (C1QA (p = 0.005); C5 (p = 0.0047), APOD (p = 0.0267); APOC3 (p = 0.0064); GP1BA (p = 0.0031); PPBP (p = 0.0465)). We further present that EV-derived fibrinogen components were paradoxically down-regulated in MI, suggesting that a compensatory mechanism may suppress post-infarct coagulation pathways, indicating potential for therapeutic targeting of this mechanism in MI. Taken together, these data demonstrated that plasma EVs contain novel diagnostic biomarkers and therapeutic targets that can be further developed for clinical use to benefit patients with coronary artery diseases (CADs). Show less

Hypertriglyceridemia results from increased production and decreased clearance of triglyceride-rich very low-density lipoproteins, a pathological condition that accounts for heightened risk of ischemic vascular diseases in obesity and type 2 diabetes. Despite its intimate association with insulin resistance, whether hypertriglyceridemia constitutes an independent risk for beta cell dysfunction in diabetes is unknown. Answering this fundamental question is stymied by the fact that hypertriglyceridemia is intertwined with hyperglycemia and insulin resistance in obese and diabetic subjects. To circumvent this limitation, we took advantage of apolipoprotein C3 (ApoC3)-transgenic mice, a model with genetic predisposition to hypertriglyceridemia. We showed that ApoC3-transgenic mice, as opposed to age/sex-matched wild-type littermates, develop hypertriglyceridemia with concomitant elevations in plasma cholesterol and non-esterified fatty acid levels. Anti-insulin and anti-glucagon dual immunohistochemistry in combination with morphometric analysis revealed that ApoC3-transgenic and wild-type littermates had similar beta cell and alpha cell masses as well as islet size and architecture. These effects correlated with similar amplitudes of glucose-stimulated insulin secretion and similar degrees of postprandial glucose excursion in ApoC3-transgenic versus wild-type littermates. Oil Red O histology did not visualize lipid infiltration into islets, correlating with the lack of ectopic triglyceride and cholesterol depositions in the pancreata of ApoC3-transgenic versus wild-type littermates. ApoC3-transgenic mice, despite persistent hypertriglyceridemia, maintained euglycemia under both fed and fasting conditions without manifestation of insulin resistance and fasting hyperinsulinemia. Thus, hypertriglyceridemia per se is not an independent risk factor for beta cell dysfunction in ApoC3 transgenic mice. Show less

One genetic cause of markedly low plasma concentrations of apolipoprotein (apo) B and low density lipoprotein (LDL)-cholesterol is familial hypobetalipoproteinemia. We aimed to determine the molecular basis for the marked hypocholesterolemia consistent with heterozygous familial hypobetalipoproteinemia in a black female subject of Xhosa lineage. Coding regions of APOB, MTTP, PCSK9,ANGPTL3, SAR1B and APOC3 were sequenced, and APOE was genotyped. COS-7 cells were transfected with plasmids containing apoB variants. Western blotting was used to detect cellular and secreted apoB, and co-immunoprecipitation performed to assess binding with the microsomal triglyceride transfer protein (MTP). Sequence analysis of the APOB gene revealed her to be heterozygous for two novel variants, c.751G>A (A224T) and c.2854G>C (V925L). She was also homozygous for the APOEε2 allele, and did not carry a PCSK9 loss-of-function mutation. Although Ala(224) is within the postulated MTP binding region in apoB, it is not conserved among mammalian species. Subsequent genotyping showed that Ala224Thr is found in a southern African population (n=654) with an allele frequency of 1.15% and is not associated with plasma lipid levels. Val(925), like Ala(224), is within the N-terminal 1000 amino acids required for lipoprotein assembly, but was not found in the population screen. However, in vitro studies showed that apoB V925L did not affect apoB48 production or secretion nor have a deleterious effect on MTP interaction with apoB. Taken together, this suggests that the hypocholesterolemia in our case may be a result of being homozygous for APOEε2 with a low baseline cholesterol. Show less

The hypothesis of the present study is that the polymorphisms in the APOC3, CEPT, ACE, and ACTN3 genes can affect the outcome of nutritional intervention and the plasma lipid profile of HIV+ patients. To test the hypothesis, genetic material was collected from buccal cells, and serum was collected for biochemical analysis. Sixty-five patients were analyzed. The incorporation of protease inhibitor (PI) was more frequent in women (77% vs 33% in men). Nutritional intervention improved anthropometric parameters independent of the genotype. Patients with the RR genotype for the ACTN3 R577X polymorphism had lower glycemia (RR = 95.4 ± 6.5 mg/dL, RX = 102.6 ± 10.6 mg/dL, XX = 110.1 ± 16.3 mg/dL; P = .03) and a greater reduction in low-density lipoproteins (LDL) after intervention (LDL: RR = -23.7 ± 15.8 mg/dL, RX = 1.32 ± 5.13 mg/dL, XX = 30.21 ± 24.4 mg/dL; P = .01). Patients using PI had a negative response to dietary intervention regarding the levels of high-density lipoprotein (-2.4 ± 1.70 with PI, 2.56 ± 1.60 mg/dL without PI; P = .02), very low density lipoprotein (0.84 ± 2.73 with IP, -5.46 ± 3.37 mg/dL without PI; P = .03), and triglycerides (1.79 ± 13.22 with PI, -34.00 ± 17.67 mg/dL without PI; P = .052). This response was also independent of the genotype (P > 0.05) and suggested the need for oral lipid-lowering drugs in all HIV+ patients using PI. Our results indicate that the ACTN3 R577X polymorphism is a good predictor of both the lipid profile and the prognosis of nutritional intervention in reducing LDL in HIV+ patients. Show less

Plasma triglyceride levels have been implicated in atherosclerosis and coronary heart disease. Apolipoprotein C-III (APOC3) plays a key role in the hydrolysis of triglyceride-rich lipoproteins to remnant particles by lipoprotein lipase (LPL) and their uptake by the liver. A rare variant in APOC3(rs138326449) has been associated with triglyceride, very low-density lipoprotein, and high-density lipoprotein levels, as well as risk of coronary heart disease. We aimed to characterize the impact of this locus across a broad set of mainly lipids-focused metabolic measures. A high-throughput serum nuclear magnetic resonance metabolomics platform was used to quantify 225 metabolic measures in 13 285 participants from 2 European population cohorts. We analyzed the effect of the APOC3 variant on the metabolic measures and used the common LPL(rs12678919) polymorphism to test for LPL-independent effects. Eighty-one metabolic measures showed evidence of association with APOC3(rs138326449). In addition to previously reported triglyceride and high-density lipoprotein associations, the variant was also associated with very low-density lipoprotein and high-density lipoprotein composition measures, other cholesterol measures, and fatty acids. Comparison of the APOC3 and LPL associations revealed that APOC3 association results for medium and very large very low-density lipoprotein composition are unlikely to be solely predictable by the action of APOC3 through LPL. We characterized the effects of the rare APOC3(rs138326449) loss of function mutation in lipoprotein metabolism, as well as the effects of LPL(rs12678919). Our results improve our understanding of the role of APOC3 in triglyceride metabolism, its LPL independent action, and the complex and correlated nature of human metabolites. Show less

Gout is associated with dyslipidaemia. Association of the apolipoprotein A1-C3-A4 gene cluster with gout has previously been reported in a small study. To investigate a possible causal role for this locus in gout, we tested the association of genetic variants from APOA1 (rs670) and APOC3 (rs5128) with gout. We studied data for 2452 controls and 2690 clinically ascertained gout cases of European and New Zealand Polynesian (Māori and Pacific) ancestry. Data were also used from the publicly available Atherosclerosis Risk in Communities study (n = 5367) and the Framingham Heart Study (n = 2984). Multivariate adjusted logistic and linear regression was used to test the association of single-nucleotide polymorphisms with gout risk, serum urate, triglyceride and high-density lipoprotein cholesterol (HDL-C). In Polynesians, the T-allele of rs670 (APOA1) increased (odds ratio, OR = 1.53, P = 4.9 × 10(-6)) and the G-allele of rs5128 (APOC3) decreased the risk of gout (OR = 0.86, P = 0.026). In Europeans, there was a strong trend to a risk effect of the T-allele for rs670 (OR = 1.11, P = 0.055), with a significant protective effect of the G-allele for rs5128 being observed after adjustment for triglycerides and HDL-C (OR = 0.81, P = 0.039). The effect at rs5128 was specific to males in both Europeans and Polynesians. Association in Polynesians was independent of any effect of rs670 and rs5128 on triglyceride and HDL-C levels. There was no evidence for association of either single-nucleotide polymorphism with serum urate levels (P ⩾ 0.10). Our data, replicating a previous study, supports the hypothesis that the apolipoprotein A1-C3-A4 gene cluster plays a causal role in gout. Show less

The association between nonalcoholic fatty liver disease (NAFLD) and apolipoprotein C3 gene (APOC3) promoter region single-nucleotide polymorphisms (SNPs) rs2854117 and rs2854116 is controversial. The aim of this study was to investigate the relationship between other polymorphisms of APOC3 and NAFLD in Chinese. Fifty-nine liver biopsy-proven NAFLD patients and 72 healthy control subjects were recruited to a cohort representing Chinese Han population. The polymorphisms in the exons and flanking regions of APOC3 and patatin-like phospholipase domain-containing protein 3 (PNPLA3) rs738409 polymorphisms were genotyped. Among the five SNPs (rs4225, rs4520, rs5128, rs2070666, and rs2070667) in APOC3, only rs2070666 (c.179 + 62 T/A) was significantly different in genotype and allele frequency (both p < 0.01) between groups of NAFLD and control. After adjusting for sex, age, serum triglycerides, total cholesterol, body mass index, and the PNPLA3 rs738409 polymorphism, the APOC3 rs2070666 A allele was an independent risk factor for NAFLD with an odds ratio (OR) of 3.683 and 95 % confidence interval (CI) of 1.037-13.084. The APOC3 rs2070666 A allele was linked to the fourth quartile of the controlled attenuation parameter values (OR 2.769, 95 % CI 1.002-7.651) in 131 subjects, and also linked to the significant histological steatosis (OR 4.986, 95 % CI 1.020-24.371), but neither to liver stiffness measurement values nor to hepatic histological activity and fibrosis in NAFLD patients. The APOC3 rs2070666 A allele is a risk factor for NAFLD independent of obesity, dyslipidemia, and PNPLA3 rs738409, and it might contribute to increased liver fat content in Chinese Han population. Show less

Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease which represents a wide spectrum of hepatic damage. Several studies have reported that NAFLD is a strong independent risk factor for coronary artery disease (CAD). And patients with NAFLD are at higher risk and suggested undergoperiodic cardiovascular risk assessment. Cardiovascular disease (CVD) is responsible for the main cause of death in patients with NAFLD, and is mostly influenced by genetic factors. Both NAFLD and CAD are heterogeneous disease. Common pathways involved in the pathogenesis of NAFLD and CAD includes insulin resistance (IR), atherogenic dyslipidemia, subclinical inflammation, oxidative stress, etc. Genomic characteristics of these two diseases have been widely studied, further research about the association of these two diseases draws attention. The gene polymorphisms of adiponectin-encoding gene (ADIPOQ), leptin receptor (LEPR), apolipoprotein C3 (APOC3), peroxisome proliferator-activated receptors (PPAR), sterol regulatory elementbinding proteins (SREBP), transmembrane 6 superfamily member 2 (TM6SF2), microsomal triglyceride transfer protein (MTTP), tumor necrosis factors-alpha (TNF-α) and manganese superoxide dismutase (MnSOD) have been reported to be related to NAFLD and CAD. In this review, we aimed to provide an overview of recent insights into the genetic basis of NAFLD and CAD. Show less

'Genetic proxies' are increasingly being used to predict the effects of drugs. We present an up-to-date overview of the use of human genetics to predict effects and adverse effects of lipid-targeting drugs. LDL cholesterol lowering variants in HMG-Coenzyme A reductase and Niemann-Pick C1-like protein 1, the targets for statins and ezetimibe, protect against ischemic heart disease (IHD). However, HMG-Coenzyme A reductase and Niemann-Pick C1-Like Protein 1-variants also increase the risk of type 2 diabetes and gallstone disease, respectively. Mutations in proprotein convertase subtilisin kexin 9 (PCSK9), apolipoprotein B, and microsomal triglyceride transfer protein cause low LDL cholesterol and protect against IHD. In addition, mutations in apolipoprotein B and microsomal triglyceride transfer protein cause hepatic steatosis, in concordance with drugs that inhibit these targets. Both mutations in PCSK9 and PCSK9-inhibition seem without adverse effects. Mutations in APOC3 cause low triglycerides and protect against IHD, and recent pharmacological APOC3-inhibition reported major reductions in plasma triglycerides. Human genetics support that low lipoprotein(a) protects against IHD, without adverse effects, and the first trial of lipoprotein(a) inhibition reduced lipoprotein(a) up to 78%. Recent genetic studies have confirmed the efficacy of statins and ezetimibe in protecting against IHD. Results from human genetics support that several lipid-lowering drugs currently under development are likely to prove efficacious in protecting against IHD, without major adverse effects. Show less

Cardiovascular and metabolic traits (CMT) are influenced by complex interactive processes including diet, lifestyle, and genetic predisposition. The present study investigated the interactions of these risk factors in relation to CMTs in the Turkish population. We applied bootstrap agglomerative hierarchical clustering and Bayesian network learning algorithms to identify the causative relationships among genes involved in different biological mechanisms (i.e., lipid metabolism, hormone metabolism, cellular detoxification, aging, and energy metabolism), lifestyle (i.e., physical activity, smoking behavior, and metropolitan residency), anthropometric traits (i.e., body mass index, body fat ratio, and waist-to-hip ratio), and dietary habits (i.e., daily intakes of macro- and micronutrients) in relation to CMTs (i.e., health conditions and blood parameters). We identified significant correlations between dietary habits (soybean and vitamin B12 intakes) and different cardiometabolic diseases that were confirmed by the Bayesian network-learning algorithm. Genetic factors contributed to these disease risks also through the pleiotropy of some genetic variants (i.e., F5 rs6025 and MTR rs180508). However, we also observed that certain genetic associations are indirect since they are due to the causative relationships among the CMTs (e.g., APOC3 rs5128 is associated with low-density lipoproteins cholesterol and, by extension, total cholesterol). Our study applied a novel approach to integrate various sources of information and dissect the complex interactive processes related to CMTs. Our data indicated that complex causative networks are present: causative relationships exist among CMTs and are affected by genetic factors (with pleiotropic and non-pleiotropic effects) and dietary habits. Show less

Elevated apoC-III levels predict increased cardiovascular risk when present on LDL and HDL particles. We developed novel high-throughput chemiluminescent ELISAs that capture apoB, lipoprotein (a) [Lp(a)], and apoA-I in plasma and then detect apoC-III on these individual lipoproteins as apoCIII-apoB, apoCIII-Lp(a), and apoCIII-apoAI complexes, respectively. We assessed the effects on these complexes of placebo or 100-300 mg volanesorsen, a generation 2.0+ antisense drug that targets apoC3 mRNA in patients with hypertriglyceridemia, including familial chylomicronemia syndrome (n = 3), volanesorsen monotherapy (n = 51), and as add-on to fibrate (n = 26), treated for 85 days and followed for 176 days. Compared with placebo, volanesorsen was associated with an 82.3 ± 11.7%, 81.3 ± 15.7%, and 80.8 ± 13.6% reduction in apoCIII-apoB, apoCIII-Lp(a), and apoCIII-apoA-I, respectively (300 mg dose;P< 0.001 for all), at day 92. Strong correlations in all assay measures were noted with total plasma apoC-III, chylomicron-apoC-III, and VLDL-apoC-III. In conclusion, novel high-throughput ELISAs were developed to detect lipoprotein-associated apoC-III, including for the first time on Lp(a). Volanesorsen uniformly lowers apoC-III on apoB-100, Lp(a), and apoA-I lipoproteins, and may be a potent agent to reduce triglycerides and cardiovascular risk mediated by apoC-III. Show less

Multiple genetic and environmental factors interact to determine an individual's predisposition to non-alcoholic fatty liver disease and its phenotypic characteristics. Association studies have found a number of alleles associated with the development of non-alcoholic steatohepatitis. Our aim was to investigate whether multiple risk-associated alleles may be present in affected monozygotic twins, indicating underlying genetic predisposition to non-alcoholic steatohepatitis. We determined the genotype of 14 candidate gene polymorphisms (at 11 unlinked loci) in a set of monozygotic twins who presented with cirrhosis within 18 months of each other. Genotyping revealed multiple single nucleotide polymorphisms at 9 independent loci in genes PNPLA3, APOC3, GCKR, TRIB1, LYPLAL1, PPP1R3B, COL13A1, and EFCAB4B, previously implicated in contributing to non-alcoholic steatohepatitis pathogenesis. In conclusion, this case series illustrates the potential cumulative effect of multiple polymorphisms in the development and potential progression of a complex trait such as NASH cirrhosis. Show less

Polymorphisms in the apolipoprotein C-III (APOC3) gene have been reported to be associated with coronary heart disease (CHD), but the data so far have been conflicting. To derive a more precise estimation of these associations, we performed a meta-analysis to investigate the three main polymorphisms (SstI, T-455C, C-482T) of APOC3 in all published studies. Databases including PubMed, Web of Science, Wanfang, SinoMed and CNKI were systematically searched. The association was assessed using odds ratios (ORs) with 95% confidence intervals (CIs). The statistical analysis was performed using Review Manager 5.3.3 and Stata 12.0. A total of 31 studies have been identified. The pooled odds ratio (OR) for the association between the APOC3 gene polymorphisms and CHD and its corresponding 95% confidence interval (95% CI) were evaluated by random or fixed effect models. A statistical association between APOC3 SstI polymorphism and CHD susceptibility was observed under an allelic contrast model (P= 0.003, OR = 1.14, 95% CI = 1.05-1.24), dominant genetic model (P= 0.01, OR = 1.14, 95% CI = 1.03-1.26), and recessive genetic model (P= 0.02, OR = 1.35, 95% CI = 1.06-1.71), respectively. A significant association between the APOC3 T-455C polymorphism and CHD was also detected under an allelic contrast (P < 0.0001, OR = 1.19, 95% CI = 1.10-1.29), dominant genetic model (P= 0.0003, OR = 1.24, 95% CI = 1.11-1.39) and recessive genetic model (P= 0.04, OR = 1.30, 95% CI = 1.01-1.67). No significant association between the APOC3 C-482T polymorphism and CHD was found under an allelic model (P= 0.94, OR = 1.00, 95% CI = 0.93-1.08), dominant genetic model (P= 0.20, OR = 1.07, 95% CI = 0.97-1.18) or recessive genetic model (P= 0.13, OR = 0.90, 95% CI = 0.79-1.03). This meta-analysis revealed that the APOC3 SstI and T-455C polymorphisms significantly increase CHD susceptibility. No significant association was observed between the APOC3 C-482T polymorphism and CHD susceptibility. Show less

Apolipoprotein C-III deficiency provides cardiovascular protection, but apolipoprotein C-III is not known to be associated with human amyloidosis. Here we report a form of amyloidosis characterized by renal insufficiency caused by a new apolipoprotein C-III variant, D25V. Despite their uremic state, the D25V-carriers exhibit low triglyceride (TG) and apolipoprotein C-III levels, and low very-low-density lipoprotein (VLDL)/high high-density lipoprotein (HDL) profile. Amyloid fibrils comprise the D25V-variant only, showing that wild-type apolipoprotein C-III does not contribute to amyloid deposition in vivo. The mutation profoundly impacts helical structure stability of D25V-variant, which is remarkably fibrillogenic under physiological conditions in vitro producing typical amyloid fibrils in its lipid-free form. D25V apolipoprotein C-III is a new human amyloidogenic protein and the first conferring cardioprotection even in the unfavourable context of renal failure, extending the evidence for an important cardiovascular protective role of apolipoprotein C-III deficiency. Thus, fibrate therapy, which reduces hepatic APOC3 transcription, may delay amyloid deposition in affected patients. Show less

Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL's size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL's content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL's content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL's ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL's APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages. Show less

Lipoprotein-associated phospholipase A2 (LpPLA2) activity was associated with higher CHD risk in a meta-analysis, which was partly dependent on circulating lipid levels. Apolipoprotein C3 loss-of-function (ApoC3 LOF) mutations were related with reduced postprandial lipemia and CHD risk. However, the association of LpPLA2 activity with ApoC3 LOF is not known. We examined the association of LpPLA2 activity and ApoC3 LOF mutations and incident cardiovascular disease (CVD) (defined as coronary heart disease [CHD] plus ischemic stroke) and all-cause mortality in the biracial longitudinal Atherosclerosis Risk In Communities (ARIC) study. The mean LpPLA2 activity was 229.3 nmol/min/mL and was higher in men and whites. LpPLA2 activity correlated positively with atherogenic dyslipidemia. ApoC3 LOF carriers had lower LpPLA2 activity levels compared to non-carriers, and there was inverse association between LpPLA2 activity and ApoC3 LOF mutations in whites. In a fully adjusted model, greater LpPLA2 activity was independently associated with incident CVD (HR 1.35, 1.09-1.68 for highest vs. lowest quintile), which was mainly explained by its association with CHD, and was also associated with all-cause mortality (HR 1.65, 1.38-1.98). Greater LpPLA2 activity was associated with increased CHD and all-cause mortality in both whites and African-Americans in the ARIC study. The inverse relation between LpPLA2 activity and ApoC3 LOF mutations suggests that delayed lipoprotein clearance may at least in part explain the observed association of LpPLA2 activity with increased CVD risk. Show less

Recent genome wide association studies have linked tribbles pseudokinase 1 (TRIB1) to the risk of coronary artery disease (CAD). Based on the observations that increased expression of TRIB1 reduces secretion of VLDL and is associated with lower plasma levels of LDL cholesterol and triglycerides, higher plasma levels of HDL cholesterol and reduced risk for myocardial infarction, we carried out a high throughput phenotypic screen based on quantitative RT-PCR assay to identify compounds that induce TRIB1 expression in human HepG2 hepatoma cells. In a screen of a collection of diversity-oriented synthesis (DOS)-derived compounds, we identified a series of benzofuran-based compounds that upregulate TRIB1 expression and phenocopy the effects of TRIB1 cDNA overexpression, as they inhibit triglyceride synthesis and apoB secretion in cells. In addition, the compounds downregulate expression of MTTP and APOC3, key components of the lipoprotein assembly pathway. However, CRISPR-Cas9 induced chromosomal disruption of the TRIB1 locus in HepG2 cells, while confirming its regulatory role in lipoprotein metabolism, demonstrated that the effects of benzofurans persist in TRIB1-null cells indicating that TRIB1 is sufficient but not necessary to transmit the effects of the drug. Remarkably, active benzofurans, as well as natural products capable of TRIB1 upregulation, also modulate hepatic cell cholesterol metabolism by elevating the expression of LDLR transcript and LDL receptor protein, while reducing the levels of PCSK9 transcript and secreted PCSK9 protein and stimulating LDL uptake. The effects of benzofurans are not masked by cholesterol depletion and are independent of the SREBP-2 regulatory circuit, indicating that these compounds represent a novel class of chemically tractable small-molecule modulators that shift cellular lipoprotein metabolism in HepG2 cells from lipogenesis to scavenging. Show less

Serum lipid levels are associated with the risk of intracerebral hemorrhage (ICH). Genetic variants in the apolipoprotein C3 (APOC3) gene were associated with plasma triglyceride (TG) and very-low-density lipoprotein (VLDL) levels. The aim of this study was to evaluate the effect of two genetic variants (1100 C/T and 3238 C/G) of APOC3 on serum lipid levels and risk of ICH. A prospective hospital-based case-control design and logistic regression analysis were utilized. We enrolled 150 ICH patients and 150 age- and gender-matched controls. The APOC3 gene polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). ICH patients had a significantly higher frequency of APOC3 3238 GG genotype [odds ratio (OR)=.97, 95% confidence interval (CI)=1.20, 7.38; P=0.02] and APOC3 3238 G allele (OR=.53, 95% CI=1.03, 2.27; P=0.04) than controls. The APOC3 3238 G allele was significantly associated with increasing plasma TG levels and VLDL levels both in ICH cases (P=0.01) and controls (P=0.02). No association was found between APOC3 1100 C/T polymorphisms and ICH. To the best of our knowledge, this is the first report in the literature that the APOC3 3238 GG genotype and G allele might contribute to an increased risk of ICH as a result of its effect on serum lipid levels. Show less

A direct relationship between high plasma triglyceride (TG) levels and increased risk of cardiovascular disease has been shown in several studies. TG are present in the blood associated with different lipoprotein classes, including hepatically-derived very low density lipoproteins (VLDL) and intestinally-derived chylomicrons. Lipoprotein lipase (LPL) is a key enzyme that hydrolyzes TG, releasing free fatty acids that accumulate in peripheral tissues and remnant lipoproteins, that are then cleared by the liver. LPL activity is finely modulated by several cofactors, including apolipoprotein C-III (apoC-III) which acts as a LPL inhibitor. The key role of apoCIII has been established in several studies: animal models lacking APOC3 gene exhibit reduced plasma TG levels, whereas the overexpression of APOC3 gene led to increased TG levels. In humans, several mutations in APOC3 gene have been identified, leading to lower apoC-III levels and associated with reduced plasma TG levels. Recently, these mutations were found to be associated with a reduced risk for cardiovascular ischemia and coronary heart disease, thus confirming the negative role of apoC-III in TG metabolism and suggesting apoC-III as possible therapeutic target for the management of hypertriglyceridemia. Show less

Genome-wide association studies have identified single-nucleotide polymorphisms (SNPs) that increase the risk of developing non-alcoholic fatty liver disease (NAFLD). One purpose of this study was to determine the frequencies of NAFLD susceptibility SNPs in a non-Hispanic white and Hispanic population who attended a clinic in northeast Albuquerque, NM. Another goal was to determine associations with selected indicators in this New Mexican population. This cohort study involving 168 volunteer subjects in the NM population (88 non-Hispanic whites, 63 Hispanics, 4 Native Americans, 11 Asian Americans, 2 unreported ethnicity). Eight SNPs within 6 NAFLD susceptibility genes including PNPLA3 (rs738409), LYPLAL1 (rs12137855), APOC3 (rs2854116, rs2854117), GCKR (rs780094, rs741038), FABP2 (rs1799883), PEMT (rs7946) were analyzed by genotyping using the TaqMan genotyping assay (Applied Biosystems, Foster City, CA). Statistical analyses were carried out using statistical package SAS 9.3. The NAFLD allele frequencies were similar in non-Hispanic whites and Hispanics except for PNPLA3 (rs738409), FABP2 (rs1799883), and PEMT (rs7946). Eight SNPs in 5 NAFLD susceptibility genes were significantly associated OR marginally associated with selected indicators for NAFLD, metabolic syndrome, overweight, obesity, insulin resistance, type 2 diabetes, hypertension, dyslipidemia. No SNPs were significantly associated with the same indicator in both the non-Hispanic white and Hispanic groups. In this population of non-Hispanic whites and Hispanics, there were only heterozygotes for the APOC3 derived alle le whereas for all other genes tested, both heterozygotes and homozygotes were found. Associations of alleles with indicators of chronic disease were different in non-Hispanic whites compared to Hispanics. Show less

Prevention or amelioration the prevalence of atherosclerosis has been an effective strategy in the management of cardiovascular diseases. The aim of the study was to scrutinize the effect of Clematichinenoside (AR) on dyslipidemia-induced atherosclerosis and explore its capability on expression of Peroxisome proliferator-activated receptor-α (PPAR-alpha), apolipoprotein A-I (APOA1) and A-II (APOA2), and suppression of apolipoprotein C-III (APOC3) genes and proteins. In the present study, we investigated atherosclerosis effect of AR using a combination of high-fat diet and balloon injury model in rabbits. The levels of biochemical indicators were evaluated in plasma, liver and HepG2 cells using immunoassay technology. In order to expose the underlying mechanism, we evaluated the regulation of PPAR-alpha, APOA1, APOA2 and APOC3 expressions by AR, and we further evaluated the interactions between them after transfection with shRNA (shPPAR-alpha) and, the action of PPAR-alpha in HepG2 cells. We could find that AR markedly promoted the PPAR-alpha transfer from cytoplasm to nucleus which resulted in the alteration of APOA1, APOA2 and APOC3 expressions in HepG2 cells. Moreover, AR significantly reduced total cholesterol, triglycerides and low-density lipoprotein cholesterol (LDL-C) levels, and elevated high-density lipoprotein cholesterol (HDL-C) level, which play an important role in dyslipidemia-induced atherosclerosis. In conclusion, AR ameliorated atherosclerosis via the regulation of hepatic lipid metabolism, and AR also contributed to the activation of PPAR-alpha, APOA1, APOA2 and APOC3. Therefore, AR could be a potential therapeutic agent in the treatment of atherosclerosis. Show less

The interaction of genetic and dietary factors, as an area of CVD research, has been explored poorly. The aim of the present study was to examine the interaction of dietary patterns and three genetic variants of APOA1 and APOC3, both independently and in combination, relative to the risk of the metabolic syndrome (MetS) in Tehranian adults. In the present matched, nested case-control study, 414 subjects with the MetS and 414 controls were selected from the participants of the Tehran Lipid and Glucose Study. Dietary patterns were determined by factor analysis. APOC3 (rs5128 3238C>G) and APOA1 (rs670, -75G>A and rs5069,+83C>T) SNP were genotyped by the conventional PCR followed by the restriction fragment length polymorphism technique. Overall, three major dietary patterns were extracted: healthy dietary pattern (HDP); Western dietary pattern (WDP); fat-sweet dietary pattern (FSDP). The A and T allele carriers of the APOA1 SNP had a greater risk of developing the MetS in the highest quartile of WDP scores (OR 3·22, 95 % CI 1·21, 8·58, P(interaction)= 0·03). Compared with other genotype combinations, the combined effect of APOC3/APOA1 (CC/GA+AA/CT+TT) genotypes showed a further increase in the risk of the MetS in the highest quartile of WDP scores (OR 1, 2·49, 8·73, 6·32, P trend< 0·001, P(interaction)= 0·003). A significant interaction was found between the quartiles of FSDP scores and the APOA1 diplotype (GA+AA/CT+TT). OR for these genotype carriers were 1, 0·65, 0·57 and 0·22 (P(trend)= 0·006) in the lowest to the highest quartile of FSDP scores when compared with the other combined genotypes (P(interaction)= 0·03). Our findings suggest that the WDP and FSDP are associated with APOA1 and APOC3 SNP in relation to the risk of the MetS. Show less

Prescription omega-3 acid ethyl esters (P-OM3) and extended release niacin (ERN) both have beneficial effects on plasma lipids and lipoproteins. The purpose of this study was to describe the effects of mono- and combination (Combo) therapy of these agents in patients with the metabolic syndrome. Very low density (VLDL), intermediate/low density (IDL/LDL, hereafter LDL), and high density lipoproteins (HDL) were isolated from 56 overweight patients with elevated triglyceride/HDL-C ratios at baseline and after 16 weeks of treatment with placebo, ERN (2g/day), P-OM3 (4g/day), or Combo and then analyzed by quantitative electrophoresis for apolipoproteins (apo) A1, A2, B, C2, C3 and E. Total plasma concentrations and the ratios of each apo with apoB (in VLDL and LDL) and with apoA1 (in HDL) were calculated. An apoC3 glycosylation index (a ratio between di- and mono-sialylated isoforms) was also determined in plasma and in each lipoprotein fraction. ERN reduced plasma apoB (-11%, p < 0.05). Combo increased LDL apoE/apoB ratio (64%, p < 0.01) and LDL apoA1/apoB (91%, p < 0.05). ERN increased the apoC3 glycosylation index only in HDL (37%, p < 0.05), whereas P-OM3 and Combo increased the index in whole plasma (48% and 49%, respectively, p < 0.05 for both) and in every lipoprotein class (VLDL: 26%, p < 0.01 and 26%, p < 0.05; LDL: 55%, p < 0.01 and 61%, p < 0.01; HDL: 43%, p < 0.001 and 44%, p < 0.001, respectively). All findings were significant after adjustment for age, sex, body mass index (BMI), smoking, medications, and baseline apo value. ERN produced a beneficial reduction in plasma apoB. The enrichment of LDL with apoE and apoA1 was unique to the Combo group and might be beneficial owing to the atheroprotective properties of apoE and HDL2 (a likely source of apoA1 in LDL fraction). The effect of therapies on the apoC3 glycosylation index is a novel finding, the implications of which will require further study. Show less

In clinical lipidology, we have focused our major efforts in defining risk status and specifying the targets of therapy by using the cholesterol content of the lipoproteins. However, we now know that these measures are variable and that they may not reveal all the valuable information that can be used to treat our patients. The amount of cholesterol in each lipoprotein can be quite different in different patients. The number of particles containing apolipoprotein B (apoB) can be abnormally high with a value for low-density lipoprotein cholesterol, which is within our guidelines. Furthermore, the content of apoC3 in apoB-containing lipoproteins can predict risk without a close association with triglycerides or cholesterol. The genome-wide association studies and studies in special families with known genetic polymorphisms have been particularly revealing relationships between these vascular risk. Show less

By detecting the variation of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) DNA methylation in preeclampsia-like mouse models generated by different ways, to explore the roles of multifactor and multiple pathways in preeclampsia pathogenesis on molecular basis. Established preeclampsia-like mouse models in different ways and divided into groups as follows: (1) Nw-nitro-L-arginine-methyl ester (L-NAME) group: wild-type pregnant mouse received subcutaneous injection of L-NAME; (2) lipopolysaccharide (LPS) group: wild-type pregnant mouse received intraperitoneal injection of LPS; (3) apolipoprotein C-III (ApoC3) group: ApoC3 transgenic pregnant mouse with dysregulated lipid metabolism received subcutaneous injection of L-NAME; (4) β2 glycoprotein I (β-2GPI) group: wild-type pregnant mouse received subcutaneous injection of β-2GPI. According to the first injection time (on day 3, 11, 16 respectively), the L-NAME, LPS and ApoC3 groups were further subdivided into: pre-implantation (PI) experimental stage, early gestation (EG) experimental stage, and late gestation (LG) experimental stage. β-2GPI group was only injected before implantation. LCHAD gene methylation levels in placental were detected in different experimental stage. Normal saline control groups were set within wild-type and ApoC3 transgenic pregnant mice simultaneously. (1) CG sites in LCHAD DNA: 45 CG sites were detected in the range of 728 bp before LCHAD gene transcription start site, the 5, 12, 13, 14, 15, 16, 19, 24, 25, 27, 28, 29, 30, 31, 32, 34, 35, 43 CG sites were complex sites which contained two or more CG sequences, others were single site which contained one CG sequence. The 3, 5, 6, 11, 13, 14, 18, 28 sites in L-NAME, LPS, ApoC3 and β-2GPI groups showed different high levels of methylation; the 16, 25, 31, 42, 44 sites showed different low levels of methylation; other 32 sites were unmethylated. (2) Comparison of LCHAD gene methylation between different groups: the methylation levels of LCAHD gene at 3, 11, 13, 14, 18 sites in L-NAME, LPS, ApoC3 and β-2GPI groups were significantly higher than those in the normal saline control group (P < 0.05); and the methylation levels of 42, 44 sites in these groups were significantly lower than those in the normal saline control group (P < 0.05). (3) Methylation of LCHAD gene at the same site between different experimental stages: ① The 3, 11, 18 sites of EG experimental stage was significantly lower than PI and LG experimental stage in L-NAME group (P < 0.05); the 3, 11, 18 sites of PI experimental stage was significantly lower than EG and LG experimental stage in LPS group (P < 0.05); these sites of PI experimental stage was significantly higher than EG and LG experimental stages in ApoC3 group (P < 0.05). ② The methylation of site 5 in L-NAME and LPS groups were significantly higher than that of the normal saline control group (P < 0.05), and the LG experimental stages were significantly higher than other stages, but in ApoC3 group, only PI and EG stages were significantly higher than the normal saline control group (P < 0.05). ③ At site 6 in L-NAME group which showed high methylation level was significantly higher than the same site in other groups which showed low methylation level (P < 0.05). ④ At 13, 14 sites, earlier preeclampsia onset caused a lower methylation level in L-NAME group, but PI experimental stage was significantly higher than EG and LG experimental stages in LPS group (P < 0.05), EG experimental stage was significantly higher than PI and LG experimental stages in ApoC3 group (P < 0.05). ⑤ At site 28, earlier preeclampsia onset caused a higher methylation level in L-NAME group, but PI experimental stage was significantly lower than EG and LG experimental stages in LPS group (P < 0.05), EG experimental stage was significantly higher than PI and LG experimental stages in ApoC3 group (P < 0.05). ⑥ The 16, 25, 31 sites in ApoC3 group were significantly higher than other groups (P < 0.05). ⑦ At site 42 in β-2GPI group was unmethylated, but it in other groups showed low methylation level, the methylation level of site 42 in β-2GPI group was significantly lower than that in other groups (P < 0.05). The methylation of 6 and 42 CG sites may be related to LCHAD gene expression in placenta of L-NAME and β-2GPI induced preeclampsia-like models respectively; LCHAD gene expression and DNA methylation may not have obvious correlation in LPS and ApoC3 induced preeclampsia-like models. Differences exist in LCHAD DNA methylation in preeclampsia-like models generated by different ways, revealed a molecular basis to expand our understanding of the multi-factorial pathogenesis of preeclampsia. Show less

Studies of the association between the apolipoprotein C3 gene (APOC3) rs5128 polymorphism and plasma levels of apolipoprotein C3 (APOC3) and lipids have reported apparently conflicting findings. This meta-analysis aimed to investigate the associations of the rs5128 polymorphism with fasting APOC3 and lipid levels. The following information was abstracted for each study: ethnicity, age, sex, health condition, sample size, genotyping and lipid assay methods, mean and standard deviation or standard error by genotypes for APOC3 and lipid variables. There were 42 eligible studies with 23846 subjects included in this meta-analysis. A dominant model was used for this meta-analysis. The results showed that the carriers of the variant allele G had higher levels of APOC3 [standardized mean difference (SMD): 0.22, 95% confidence interval (CI): 0.12-0.31, P<0.00001], triglycerides (TG) (SMD: 0.33, 95% CI: 0.23-0.44, P<0.00001), total cholesterol (TC) (SMD: 0.15, 95% CI: 0.09-0.22, P<0.00001), and low-density lipoprotein cholesterol (LDL-C) (SMD: 0.11, 95% CI: 0.04-0.17, P=0.001) than the non-carriers. No significant association between the APOC3 rs5128 polymorphism and lower levels of high-density lipoprotein cholesterol (HDL-C) was detected under the dominant model (SMD: -0.03, 95% CI: -0.06-0.01, P=0.156). The results from the present meta-analysis demonstrate a significant association between the APOC3 rs5128 polymorphism and higher levels of APOC3, TG, TC and LDL-C, but further studies are needed to elucidate the underlying mechanisms. Show less

A typical human exome harbors dozens of loss-of-function (LOF) variants, which can lower disease risk factor levels and affect drug efficacy. We hypothesized that LOF variants are enriched in genes influencing risk factor levels and the onset of common chronic diseases, such as cardiovascular disease and diabetes. To test this hypothesis, we sequenced the exomes of 8,554 individuals and analyzed the effects of predicted LOF variants on 20 chronic disease risk factor phenotypes. Analysis of this sample as discovery and replication strata of equal size verified two relationships in well-studied genes (PCSK9 and APOC3) and identified eight new loci. Previously unknown relationships included elevated fasting glucose in carriers of heterozygous LOF variation in TXNDC5, which encodes a biomarker for type 1 diabetes progression, and apparent recessive effects of C1QTNF8 on serum magnesium levels. These data demonstrate the utility of functional-variant annotation within a large sample of deeply phenotyped individuals for gene discovery. Show less