Diverse studies have proposed an important role of the APOE in hypercholesterolemia and statin response based in this association with APOE ε2/ε3/ε4 genotypes, but little information is known about the relationship of these variables with the expression status of APOE. Here, we describe the mRNA expression profile in PBMC from NL individuals and HC patients treated with atorvastatin.
Absence or structural mutations of APOE cause significant disorders in lipid metabolism and cardiovascular diseases. Deficiency of apoE results in massive accumulation of remnant lipoproteins, leading to severe hypercholesterolemia and atherosclerosis in human and apoE knockout mice . Here, we reported that HC individuals have lower APOE mRNA expression than NL individuals, which is concordant with the previous information related to apoE deficiency.
The APOE mRNA expression is extremely complex with regulation in a tissue-specific manner and in response to cellular changes and extra and intra-cellular factors . The expression of cholesterol acceptors in the efflux process such as apoAI, as well as transporter proteins involved in this process have been described to activate APOE transcription in human adipocytes and macrophages [9, 10]. Accordingly, we observed that individuals with low expression of APOE present a decreased apoAI plasma concentration. Moreover, we also reported that these individuals have increased concentrations of total and LDL cholesterol and apoB. The relation of plasma levels of LDL cholesterol with APOE expression in PBMC was previously reported in children with obesity . The increased concentrations of particles which depend on the LDL receptor (LDLR) for their clearance from plasma is consistent with the key role of apoE as a high affinity ligand for the LDLR in the cholesterol homeostasis .
APOE allele frequencies have demonstrated to be heterogeneous among different populations, but the ε3 allele is almost invariably the most common and ε2 the rarest allele . In this study, the frequencies of APOE alleles observed in the overall population (ε2: 5%, ε3: 79% and ε4:16%) were similar to earlier studies in European descendant population [13, 14], African American  and Brazilian populations .
Several genetic factors have been related to hypercholesterolemia, however in most of the cases the contribution of these genetic factors to the risk for hypercholesterolemia depends on environmental factors. Our results showed differences in genotype and allele frequencies between normolipidemic and hypercholesterolemic groups, suggesting that APOE ε2 allele confers protection against hypercholesterolemia. This characteristic persists even after adjustment for covariates that have been largely associated with hypercholestolemia, such as gender, ethnics, history of CAD, age, hypertension, obesity, cigarette smoking and physical activity, suggesting that APOE ε2 could be considered an independent factor that protect against hypercholesterolemia in our sample population.
Ferreira and co-workers  did not found differences in APOE ε2/ε3/ε4 genotypes between normolipidemic and dyslipidemic Brazilian individuals. On the other hand, and in line with our results, it has been reported higher frequency of ε2 allele in normolipidemic than hypercholesterolemic individuals from South America . Moreover, a previous study in the Brazilian population reported that, compared with the ε2 allele, the presence of ε3 allele increases more than two times the risk for dyslipidemia (OR: 2.31, CI:1.06-5.06) , which is in agreement with our results.
The effects of APOE polymorphisms on plasma lipids have been described by several studies and the evidence suggests that APOE ε2 is associated with lower, whereas ε4 with higher, concentrations of plasma total cholesterol, LDL cholesterol and apoB in comparison with the ε3 allele . We reported a less atherogenic lipid profile of ε2 allele, as well as a contribution of ε4 allele for higher total and LDL cholesterol and apoB in normolipidemic individuals. However, the association between APOE polymorphisms and plasma lipids were detected exclusively in the normolipidemic group. In agreement with this characteristic, an association of APOE genotypes with basal plasma lipids in normolipidemic individuals, but not in dyslipidemic patients, was previously reported in our population . The authors described that ε2 allele carriers had significantly lower total, LDL and non-HDL cholesterol compared to ε3 and ε4 allele carriers only in normolipidemic individuals. Moreover, other studies were not able to demonstrate any association between APOE ε2/ε3/ε4 genotypes and total and LDL cholesterol in patients with familial hypercholestolemia  and polygenic dyslipidemia [21, 22]. Nerveless, the lack of association of APOE polymorphisms with plasma lipids in hypercholesterolemic patients in our sample seems to be attributable to the small number of individuals carrying the ε2 allele that could be considered an important limitation of our study.
The variation on plasma lipids according to APOE ε2/ε3/ε4 genotypes are believed to stem mainly from structural and biophysical properties of apoE isoforms . ApoE4-containing lipoproteins exhibit a high binding ability to their receptors that cause a more efficient catabolism and an accelerated clearance of chylomicrons and VLDL-remnants, leading to down regulation of LDLR and HMGCR and to increased LDL cholesterol levels in plasma. On the contrary, lipoproteins containing the apoE2 isoform present lower affinity compared to apoE4 and apoE3 isoforms that result in decreased cholesterol levels.
In the present study, no differences were observed in the change of lipid levels in response to atorvastatin treatment according to APOE genotypes. Although many studies have evaluated the influence of APOE polymorphism on statin response, some of these studies had controversial results. Whereas there is a strong line of evidence linking APOE ε2/ε3/ε4 genotypes with the efficacy of statin treatment [23–26], other studies did not reveal any association between APOE genotypes and response to treatments with various statins [27–29]. Commonly, evidence supports that APOE ε3 allele is associated with better response than ε4 allele in term of LDL cholesterol decrease and, in addition, individuals carrying the ε2 allele have greater reduction of LDL cholesterol than ε3 homozygotes . These differences result from the improved activity of HMGCR in ε2 compared to ε3 allele carriers due to the modulation of intracellular cholesterol by the upregulation of hepatic LDLR, which has lower affinity for the apoE2 isoform that results in an improved response of ε2 allele carriers to the inhibition of HMGCR by statins. On the other hand, the LDLR presents higher affinity for apoE4 isoform and the effect of statin therapy is diminished in ε4 allele carriers when compared to ε2 or ε3 .
In the last years, GWAS have provided new perspectives and a more comprehensive approach for identifying genetic loci associated to statin response. Thompson et al. (2009), using a platform of 291, 988 SNPs did not observe any association between genotypes and atorvastatin response at beginning, when 1, 984 individuals were analyzed, however further analysis in 5745 individuals from the Treating to New Target (TNT) trial using a candidate gene approach reported a strong association between APOE ε2/ε3/ε4 genotypes and LDL cholesterol statin response . Furthermore, a recent study has evaluated the response to diverse statins using a GWAS approach involving nearly 4, 000 individuals from three different trials of statin efficacy [Cholesterol and Pharmacogenetics (simvastatin), Pravastatin/Inflammation CRP evaluation (pravastatin) and TNT (atorvastatin)] . The authors did not found any association between APOE SNPs and statin response, however the SNP rs4429638, located in the APOC1 gene and near APOE, was associated with change LDL cholesterol suggesting a possible involvement of APOE locus in statin efficacy.
Despite the number of studies investigating the response to statins according to APOE genotypes, the effect of HMGCR inhibitors on apoE protein and mRNA expression has been poorly studied, particularly using in vivo models. Atorvastatin and cerivastatin demonstrated to reduce apoE protein secretion and APOE mRNA expression in THP-1 derived macrophages after 24h of treatment in a dose dependent manner . Conversely, in cultured human monocyte-derived macrophages, lovastatin increased APOE mRNA levels but decreased apoE secretion , phenomena that the authors attributed to the increase of apoE not destined for secretion. On the other hand, regarding in vivo studies, Guan et al. reported that APOE mRNA levels in mononuclear cells of hyperlipidemic diabetic patients taking simvastatin (5-10 mg/day) did not differ from those without statin treatment . These contradictory results from in vitro experiments and the data reported for Guan and co-workers and our observations in PBMC from hypercholesterolemic individuals (not change after 10 mg/day atorvastatin treatment) could be explained by the differences in the cellular models used by the authors. Moreover, we observed that patients without LDL cholesterol goal achievement had lower APOE mRNA expression that could suggest a possible involvement of the modulation of this gene in the statin response.