Low-Expression Variant of Fatty Acid–Binding Protein 4 Favors Reduced Manifestations of Atherosclerotic Disease and Increased Plaque StabilityCLINICAL PERSPECTIVE
Background—Fatty acid–binding protein 4 (FABP4 or aP2 in mice) has been identified as a key regulator of core aspects of cardiometabolic disorders, including lipotoxic endoplasmic reticulum stress in macrophages. A functional promoter polymorphism (rs77878271) of human FABP4 gene has been described resulting in reduced FABP4 transcription.
Methods and Results—We investigated the effects of this low-expression variant of FABP4 on cardiovascular morbidity and carotid atherosclerosis on a population level (n=7491) and in patient cohorts representing endarterectomized patients with advanced carotid atherosclerosis (n=92) and myocardial infarction (n=3432). We found that the low-expression variant was associated with decreased total cholesterol levels (P=0.006) with the largest reduction in variant allele homozygotes. Obese variant allele carriers also showed reduced carotid intima-media thickness (P=0.010) and lower prevalence of carotid plaques (P=0.060). Consistently, the variant allele homozygotes showed 8-fold lower odds for myocardial infarction (P=0.019; odds ratio, 0.12; 95% confidence interval, 0.003–0.801). Within the carotid plaques, the variant allele was associated with a 3.8-fold reduction in FABP4 transcription (P=0.049) and 2.7-fold reduction in apoptosis (activated caspase 3; P=0.043). Furthermore, the variant allele was enriched to patients with asymptomatic carotid stenosis (P=0.038). High FABP4 expression in the carotid plaques was associated with lipid accumulation, intraplaque hemorrhages, plaque ulcerations, and phosphoactivated endoplasmic reticulum stress markers.
Conclusions—Our results reveal FABP4 rs77878271 as a novel variant affecting serum total cholesterol levels and cardiovascular risk. A therapeutic regimen reducing FABP4 expression within the atherosclerotic plaque may promote lesion stability through modulation of endoplasmic reticulum stress signaling, and attenuation of apoptosis, lipid burden, and inflammation.
- carotid stenosis
- coronary artery disease
- ER stress
- FABP4 protein, human
Identification of novel therapeutic targets and design of effective prevention strategies in cardiometabolic diseases require the understanding of the molecular pathways linking metabolic perturbations to inflammatory responses, especially in the vascular wall. One potential molecular link integrating metabolic and inflammatory signals is fatty acid–binding protein 4 (FABP4; adipocyte-FABP or aP2 in mice), a lipid chaperone highly expressed both in adipocytes and in macrophages.1–9 In mice, FABP4 deficiency or pharmacological inhibition of FABP4 protein with an orally active small-molecule protected against atherosclerosis5,6,10,11 and reduced obesity-related metabolic disorders and type 2 diabetes mellitus.1,6,12,13 The proatherogenic effects of FABP4 in atherosclerosis have been attributed almost solely to its actions in macrophages.5,10,11 Specifically, FABP4 regulates the unfolded protein response (UPR),5 an adaptive program of the cell employed to protect against increased endoplasmic reticulum (ER) stress.14 In the macrophages of the vascular wall ER stress may arise following the accumulation of free cholesterol into the ER, which distorts its function and activates the UPR pathways to recover ER functionality and to divert unsalvageable cells to apoptosis.15,16 In human atherosclerosis, increased ER stress has been reported in vulnerable coronary plaques in connection with acute coronary syndrome.17
Clinical Perspective on p 598
High expression of FABP4 in atherosclerotic carotid plaques (CPs) has been linked with stroke-prone carotid atherosclerosis.18–21 Interestingly, a functional polymorphism rs77878271 (T-87C) was recently described in the promoter of human FABP4 gene.22 This polymorphism disrupts a CAAT box/enhancer-binding protein α binding sequence and results in reduced transcription of FABP4 gene both in vitro and in adipose tissue of carriers in vivo.22 In that study, this low-expression variant of FABP4 was associated with reduced risk of coronary heart disease.22 The same study also demonstrated that the low-expression variant reduced the risk for hypertriglyceridemia and obesity-related type II diabetes mellitus in man.
We previously discovered FABP4 as one of the most upregulated genes in unstable, stroke-associated CPs when compared with clinically silent asymptomatic CPs.18 Although a proposed antiatherogenic mechanism of FABP4 deficiency has been dissected in mice and in in vitro studies, the evidence linking FABP4 to human atherosclerosis is largely missing. Because the promoter variant rs77878271 has been shown to reduce FABP4 expression, we considered this low-expression variant of FABP4 as a naturally occurring genetic model of reduced FABP4 expression in man. We could establish that the variant also decreased FABP4 expression in human atherosclerotic lesions. This enabled us to investigate the pathophysiological mechanisms of FABP4 expression in atheroma from the development of early stage atherosclerosis to its effects on lipid accumulation, ER stress, and apoptosis in advanced vulnerable lesions and associated atherothrombotic diseases on a population level.
The Health 2000 Survey
The Health 2000 was a large epidemiological health survey performed in Finland from fall 2000 to spring 200123 and included 8028 participants representing the Finnish population aged ≥30 years. In-depth cardiovascular examinations were performed in the cardiovascular disease and diabetes subcohort (SVT-D, sample size 1867 and participation rate 82%), the participants of which were aged 45 to 74 years and living within the catchment areas of 5 Finnish University Hospitals. Detailed information on clinical examination and disease definitions are provided in the Data Supplement. Carotid ultrasound examinations were performed by high-resolution B-mode equipment as described.24 The reported carotid intima-media thickness (IMT) represents the mean of the common carotid artery IMT and internal carotid IMT. Altogether 1448 genotyped individuals with available carotid ultrasound data participated in the study.
The Helsinki Carotid Endarterectomy Study
Patients with advanced carotid stenosis belong to The Helsinki Carotid Endarterectomy study (HeCES).18,25–27 Briefly, the study included 92 consecutive patients with a high-grade carotid stenosis (>70%)28 who underwent carotid surgery in Helsinki University Central Hospital during 1997 to 2000. All patients underwent neurological examination, carotid ultrasound and preoperative digital subtraction angiography, brain imaging, and blood sampling for DNA and serum. Seventy-four patients had a history of transient ischemic attack or stroke from carotid stenosis (symptomatic patients). Eighteen patients had never experienced cerebrovascular symptoms or had a history of stroke or transient ischemic attack of other cause (asymptomatic patients). In addition, quantitative real-time reverse transcriptase-polymerase chain reaction (qPCR) and some immunohistochemical analyses were performed in a subgroup of patients with a clinically defined ischemic stroke or retinal infarct in the ipsilateral carotid territory (n=25) and asymptomatic patients (n=18).
The Corogene Study
The Corogene study included 5295 consecutive Finnish patients assigned to coronary angiogram in 4 hospitals servicing 1.5 million people in the Hospital District of Helsinki and Uusimaa. Of the Corogene study, 2500 patients with acute coronary syndrome (International Classification of Diseases-Tenth Revision: I20–I25) were included in a genome-wide association study with a case–control setting.29 Here, we used available genome-wide association data to test the association of rs77878271 with myocardial infarction (MI, International Classification of Diseases-Tenth Revision, I21) using 1565 MI cases from the Corogene cohort and 1867 controls selected from the FINRISK surveys.30
The FABP4 rs77878271 polymorphism was genotyped either by TaqMan SNP Genotyping Assay (Applied Biosystems), by sequencing, or by Illumina 660K BeadChip array and imputational approaches (in the Data Supplement).
The CPs were removed en bloc in endarterectomy, rinsed with saline, and graded macroscopically. All plaques represented complicated American Heart Association-class VI lesions.31 The plaques were cut into 5 longitudinal segments, each containing a portion of the tightest stenosis and destined for a specific purpose: histological and immunohistochemical examinations or RNA and protein extraction as previously described.18,25–27
qPCR was performed using Assays-on-Demand Gene Expression Products and ABI PRISM 7000 Sequence Detection System (Applied Biosystems) as described.18
Protein Isolation and FABP4 ELISA
Total cellular proteins were isolated from the carotid specimens using the Trizol-reagent. FABP4 protein was quantified using a human AFABP ELISA kit (BioVendor) and expressed as picograms of FABP4 per microgram of total protein.
Histology and Immunohistochemistry
Plaque neutral lipids were stained using Oil-red-O (ORO) and ferric iron using Perls’ Prussian blue stainings. Adjacent sections were used in the stainings against CD36 and ATP-binding cassette transporter A1 (ABCA1),27 as well as for colocalization of tissue iron, FABP4, and phosphoactivated ER stress markers. Details of immunostainings are given in the Data Supplement. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) analysis was performed using In situ Cell Death Detection Kit (Roche). Nonspecific IgGs or PBS served as negative controls.
Light microscopy (Axioplan 2, Carl Zeiss) was performed by investigators blinded to the clinical data. Histological and immunohistochemical stainings were graded by eye, apart from the stainings against activated caspase 3 (aCASP3) where the amount of immunoreactive cells was counted by eye and divided by the total area of the specimen giving a relative density for aCASP3 immunoreactive cells.26
Differences in continuous variables were tested by Mann–Whitney U, Kruskal–Wallis, or independent samples t test when indicated and between nominal variables by χ2, Jonckheere–Terpstra, or Fisher's exact test. Bivariate correlations were analyzed by Spearman correlation (rs). P values ≤0.05 were considered significant. Data are expressed as mean±SE unless otherwise indicated. All statistical analyses were performed using IBM SPSS Statistics version 19 unless otherwise indicated.
The association of FABP4 rs77878271 with serum total and low-density lipoprotein-cholesterol (total-C and LDL-C) levels and apolipoprotein B (apoB) concentration was examined using linear regression adjusting for age, sex, body mass index (BMI), the use of lipid-lowering medication, and FABP4 rs77878271 genotypes. Carotid IMT was modeled using linear regression adjusting the effect of rs77878271 genotype for age, sex, high-density lipoprotein-cholesterol, apoB, triglycerides, fasting blood glucose, systolic blood pressure, and smoking. In addition, an interaction between rs77878271 genotype and obesity was included in the model. Variables entered in the models were checked for multicollinearity, and all tolerance values were >0.48 indicating no serious collinearity between explanatory variables.
The genetic association of rs77878271 with MI was tested in Health 2000 (cases, n=532; noncases, n=6959) and Corogene (cases, n=1565; controls, n=1867) data sets separately and by meta-analysis of these 2 cohorts. In these analysis, the matched case–control setting was not used. The association was tested with logistic regression for additive model and the exact Mantel–Haenszel test for the recessive model (as the number of homozygous MI cases was small, n=1) using R (version 2.15.0). Logistic regression model was adjusted for age, sex, and BMI. The association of FABP4 rs77878271 with incident ischemic cardiovascular events was analyzed in the Health 2000 cohort using Cox proportional hazards regression with age as the time scale. End point data were gathered using hospital discharge and causes of death registers from 1971 to 2011. The model was adjusted for sex and rs77878271 genotypes.
Study protocols of the Health 2000, HeCES, and Corogene studies were approved by appropriate Ethics Committees of the Helsinki and Uusimaa Hospital region, and all participants gave signed informed consent.
Prevalence of the Low-Expression Variant in the Finnish Population
Genotyping of the FABP4 rs77878271 promoter polymorphism was successful in 7491 individuals (99% of available DNA samples) of the Health 2000 Survey. The genotype frequencies were 88.7% (TT), 10.9% (TC), and 0.4% (CC; Table 1). The minor allele frequency was 5.8%. The values followed Hardy–Weinberg equilibrium (P=0.58).
Low-Expression Variant of FABP4 Associates With Reduced Serum Total Cholesterol Levels
There were no significant differences in the baseline characteristics and cardiovascular risk factors between FABP4 rs77878271 genotypes (Table I in the Data Supplement). After adjusting for age, sex, BMI, and the use of lipid-lowering medication the FABP4 rs77878271 variant allele demonstrated a significant dose-dependent association with lower serum total-C levels (P=0.006; n=6868), with consistent trends toward lower LDL-C (P=0.066; n=6840) and apoB (P=0.070; n=6868) concentration (Table II in the Data Supplement). When compared with major allele homozygotes (TT), TC heterozygotes showed significantly reduced serum total-C (P=0.020; Figure 1A). The effect in TC heterozygotes and in the variant allele homozygotes (CC homozygotes) on serum total-C were ≈−2% and −6%, respectively. The effect was largest in CC homozygotes, with the foremost reduction in apoB levels (P=0.068; Figure 1A). No significant associations with FABP4 rs77878271 and apolipoprotein A-I concentration or high-density lipoprotein-cholesterol levels were detected (data not shown).
Because aP2 deficiency (aP2−/−) in mice is associated with reduced circulating cholesterol levels selectively in obesity,13 we examined whether body weight would have similar modulatory effect on this association in man. There was a clear enhancement in the reduction of total and LDL-C levels and apoB concentration in CC homozygotes in the highest BMI quartile (Q4; BMI>29.5; Figure 1B–1D). In the regression analysis of BMI Q4 alone, CC homozygotes showed substantial reduction in serum total-C (≈−16%; P=0.034) and apoB (≈−20%; P=0.018) levels when compared with TT homozygotes (Figure 1E).
Low-Expression Variant of FABP4 Associates With Lower Carotid Artery IMT and Lower Prevalence of CPs in Obese Carriers
Next we studied, whether the low-expression variant was associated with carotid IMT or the frequency of CPs, early surrogate markers of cardiovascular risk. The mean IMT in the SVT-D subcohort was 0.94±0.23 mm (Table III in the Data Supplement). There were no significant differences in the mean IMT values between genotypes (0.94±0.23 mm in TT, 0.93±0.22 mm in TC, and 0.97±0.32 mm in CC) or between T and C allele carriers. Similarly, no differences were seen in the frequency of CPs (15.9% in TT, 13.0% in TC, and 14.3% in CC).
Studies in aP2−/− mice suggest that the effects of aP2 deficiency in vivo manifest under cumulative metabolic strain (ie, in obesity).1,13 Therefore, we stratified the SVT-D subcohort according to obesity (BMI≥30; Table IV in the Data Supplement). In obese individuals, the low-expression variant was associated with a significantly lower carotid IMT (0.98±0.22 mm in TT versus 0.90±0.16 mm in TC/CC; P=0.010), as well as a 3.4-fold reduction in the prevalence of CPs (17.1% versus 5.1%; P=0.060; Table 2). These findings were consistent in both sexes although the reduction in CPs was most prominent in obese women (CP prevalence’s were 16.6% in TT versus 0.0% TC/CC; P=0.029; Table 2).
In multivariate analysis of the whole SVT-D subcohort adjusting for known risk factors for atherosclerosis (age, sex, high-density lipoprotein-cholesterol, apoB, triglycerides, fasting blood glucose, systolic blood pressure, and smoking), the low-expression variant or obesity alone had no significant effect on carotid IMT. However, if we allow for the interaction between the low-expression variant and obesity, it is significant (P=0.021; Table V in the Data Supplement), suggesting that the obese carriers of the low-expression variant have significantly lower carotid IMT when compared with obese TT homozygotes.
Homozygote Carriers of the Low-Expression Variant Show Low Rate of Cardiovascular Events
In the Health 2000 Survey, there were 28 individuals homozygous for the low-expression variant of FABP4 (CC). At the baseline health examination, they had no history of ischemic strokes (0% versus 3.4% in TT and 3.2% in TC genotype) or MIs (0% versus 5.1% in TT and 5.0% in TC genotype; Table 1). Similarly, during a 10-year follow-up, there were no new cases of MIs or major coronary disease events among the CC homozygotes (0% versus 2.5% and 5.2% in TT and 2.8% and 5.0% in TC genotype, respectively). One ischemic stroke occurred among the CC homozygotes. The cumulative prevalences were 0% in CC homozygotes versus 11.8% in TT and 11.3% in TC genotype for major coronary disease events (TT/TC versus CC; P=0.109) and 3.6% in CC homozygotes when compared with 16.6% in TT and 15.6% in TC genotype for all major ischemic cardiovascular events (TT/TC versus CC; P=0.073; Table 1). Similarly, in Cox regression analysis, the hazard ratio for incident ischemic cardiovascular events for CC homozygotes was 0.33 (TT versus CC; 95% confidence interval [CI], 0.05 to 2.35; P=0.268) and for TC heterozygotes 0.90 (TT versus TC; 95% CI, 0.73 to 1.10; P=0.264; Figure I in the Data Supplement).
We further explored the association of rs77878271 with MI in a large case–control data set, derived from the Corogene study,29 with available genome-wide genotyping data using imputational approaches. In this data set, using a recessive model, the rs77878271 CC homozygotes showed 6-fold lower odds for MI (P=0.08; odds ratio, 0.17; 95% CI, 0.004–1.33; MI, n=1565; controls, n=1867; imputation info 0.97; minor allele frequency 5.9%; Hardy–Weinberg equilibrium from best guess genotypes P=0.90). In a pooled analysis of Health 2000 and Corogene data sets, using a recessive model, the rs77878271 CC homozygotes showed 8-fold lower odds for MI (P=0.019; odds ratio, 0.12; 95% CI, 0.003–0.801, MI, n=2097; controls, n=8826). Rs77878271 was not associated with MI using an additive model and logistic regression (P=0.41).
Low-Expression Variant of FABP4 Associates With Stable Carotid Stenosis
We next analyzed the low-expression variant of FABP4 in the HeCES cohort consisting of patients with advanced (>70%)28 carotid atherosclerosis25 (n=92; Table VI in the Data Supplement). Among patients with advanced carotid atherosclerosis, 12.1% were heterozygote carriers of the low-expression variant with a minor allele frequency of 6.0%, which did not differ from population controls (Table VII in the Data Supplement). Genotype frequencies followed Hardy–Weinberg equilibrium (P=0.54). However, patients with stable, clinically asymptomatic carotid atherosclerosis demonstrated higher than expected heterozygosity frequency of the low-expression variant when compared with symptomatic patients (27.8% versus 8.2%; P=0.038; Table VII in the Data Supplement). In addition, patients with asymptomatic carotid disease showed a difference in allele distribution when compared with the Health 2000 survey population controls (P=0.045).
FABP4 Expression Is Elevated in Unstable CPs and Reduced in the Carriers of the Low-Expression Variant
Previously, we identified significant overexpression of FABP4 mRNA in stroke-associated when compared with asymptomatic CPs in a genome-wide analysis.18 A 2.2-fold overexpression was confirmed using qPCR in stroke-associated when compared with asymptomatic CPs (1.95±0.37 versus 0.90±0.32; P=0.015; Figure 2A). In line with the qPCR experiment, FABP4 protein quantification by ELISA from the same CPs revealed a marked 3.9-fold higher level of FABP4 protein in the stroke-associated when compared with asymptomatic CPs (30.2±8.0 versus 7.7±1.5 pg/μg of total protein; P=0.003; Figure 2B). FABP4 protein levels and mRNA expression in the CPs were highly correlated (rS=0.695; P<0.001 not shown). Because the low-expression variant carriers display reduced FABP4 transcription in adipose tissue,22 we examined whether a similar effect could be observed in endarterectomized CPs. Patients carrying the low-expression variant of FABP4 showed a 3.8-fold reduction in FABP4 mRNA expression (0.44±0.20 versus 1.68±0.30; P=0.049) and a trend toward lower FABP4 protein levels (6.2±1.6 versus 22.5±5.5 pg/μg of total protein; P=0.107) in their CPs when compared with TT homozygotes (Figure 2C and 2D).
FABP4 Expression Associates With Lipid and Macrophage Accumulation Into Carotid Atheroma
Animal studies have suggested that FABP4 deficiency promotes cholesterol efflux and resistance toward cholesterol accumulation in macrophages.2,5 However, the relationship of FABP4 expression with lipid accumulation and inflammation in carotid atheroma has not been studied. We analyzed the relationship between intra- and extracellular neutral lipid accumulation (ORO) and FABP4 expression in the CPs. We found that FABP4 expression was correlated with the degree of carotid stenosis (mRNA, rS=0.334; P=0.037 and protein, rS=0.521; P=0.001, not shown). In the CPs, increasing FABP4 immunoreactivity was highly correlated with increasing HAM56 immunoreactive area detecting macrophages (P=0.008; Figure II in the Data Supplement). Increasing FABP4 immunoreactivity correlated with increasing intra- and extracellular ORO staining grades (P=0.031 and P=0.002, respectively; Figure 3A–3C). In part, cellular lipid accumulation in atheroma is regulated by CD36-mediated LDL uptake in proportion to balancing efflux through ABCA1 and ATP-binding casette transporter G1 (ABCG1). We found that FABP4 mRNA expression and protein levels were correlated with CD36 mRNA levels (rS=0.863; P<0.001 for mRNA expression and rS=0.786; P<0.001 for protein levels, respectively, not shown) and with immunoreactive area of CD36 protein (P=0.110 and P=0.034, respectively; Figure 3D and 3E). This association could not be demonstrated between FABP4 expression and ABCA1 immunoreactivity. However, FABP4 protein levels were correlated with the ratio of CD36/ABCA1 immunoreactivity27 (rS=0.595; P=0.002, not shown). FABP4 protein levels were 4.5-fold higher in those CPs where the immunoreactivity for CD36 exceeded that for ABCA1 (55.8±18.3 versus 12.3±3.66 pg/μg of total protein; P=0.009; Figure 3F).
Plaque Ulcerations and Intraplaque Hemorrhages Associate With FABP4 Expression
Macroscopic ulcerations are strongly associated with symptomatic carotid disease because they indicate a recent thromboembolic event.32 We discovered a 2.8-fold upregulation of FABP4 mRNA (2.2±0.44 versus 0.78±0.24; P=0.005) and a 4.4-fold elevation of FABP4 protein (32.5±9.4 versus 7.4±1.1 pg/μg of total protein; P=0.001) in CPs with macroscopic ulcerations when compared with nonulcerated CPs (Figure 4A and 4B). Moreover, CPs that were both ulcerated and ipsilateral to a carotid territory cerebral infarction had 4-fold higher FABP4 protein levels when compared with ulcerated plaques in asymptomatic patients (41.0±12.2 versus 10.2±4.4 pg/μg of total protein, respectively; P=0.046; Figure 4C). FABP4 immunoreactivity was also increased in CPs with intraplaque hemorrhages (IPHs) when compared with CPs without IPHs (P<0.001; Figure 4D). Frequent IPHs promote in situ accumulation of remnant tissue iron that may further provoke oxidative stress, another recognized atheromatous UPR activator.33 We found a striking colocalization between FABP4 immunoreactivity and tissue iron (Prussian blue staining), the surrogate marker of previous IPHs (Figure 5F). Consistently, there was a strong association between Prussian blue staining and FABP4 immunoreactivity (P<0.001; Figure 4E). Increasing FABP4 immunoreactivity was also highly correlated with platelet (CD42b; P=0.001) and with fibrin immunoreactivity (P<0.001), both distinctive for more recent IPHs (Figure 4F and 4G). Because the atherogenic effects of FABP4 in the atheroma are almost completely mediated via macrophage functions,5,10,11 we investigated the connection between FABP4 expression and CD163-mediated macrophage hemoglobin scavenging in the CPs and found that the expression of FABP4 and CD163 mRNA (rS=0.828; P<0.001, not shown) and their immunoreactivities (P<0.001; Figure 4H) were correlated. The carriers of the low-expression variant also had 1.6-fold lower CD163 mRNA expression in their CPs when compared with major allele homozygotes (1.33±0.09 versus 0.81±0.19, respectively; P=0.022; Figure 4I), despite equal frequency of IPHs in both genotypes (45% versus 52%, respectively).
FABP4 Expression Associates With ER Stress in Carotid Atheroma
In mice, aP2−/− has been shown to reduce macrophage apoptosis in atherosclerotic lesions in vivo through alleviation of lipotoxic ER stress.5 The low-expression variant of FABP4 enables similar investigations on the potential relation between FABP4 expression and chronic ER stress in advanced CPs. For this purpose, we quantified apoptosis markers aCASP3 and TUNEL reactivity in the CPs. We found that increasing FABP4 immunoreactivity was associated with higher relative density of aCASP3 immunoreactive cells and TUNEL reactivity in the CPs (P=0.019 and P=0.008, respectively; Figure 5A and 5B). This association was also evident between FABP4 protein levels in the CPs and the relative density of aCASP3 immunoreactive cells and TUNEL reactivity (rS=0.487; P=0.003 and P=0.023, respectively; Figure IIIA and IIIB in the Data Supplement). The relative density of aCASP3 immunoreactive cells and TUNEL reactivities also strongly associated with increasing intracellular ORO staining grades (P=0.003 and P=0.006, respectively; Figure IIIC and IIID in the Data Supplement). In line, FABP4 immunoreactivity, intracellular ORO staining grades, and the relative density of aCASP3 immunoreactive cells were positively associated with each other (Figure 5C). In the low-expression variant carriers, the relative density of aCASP3 immunoreactive cells was reduced by 2.7-fold (1.10±0.37 versus 3.00±0.60; P=0.043), and a trend toward less TUNEL reactivity was observed when compared with TT homozygotes (P=0.095; Figure 5D and 5E). Immunohistochemical stainings of FABP4 together with successively phosphoactivated ER stress markers, phosphorylated pancreatic ER kinase and phosphorylated eukaryotic translation initiation factor 2 alpha, and apoptosis marker, aCASP3 from consecutive CP sections, revealed the presence of these UPR markers with apoptosis in macrophage-rich areas of the CPs (Figure 5F).
Genetic models and in vitro studies have outlined a crucial role for aP2, the mouse orthologue of human FABP4, in the crossroads of metabolic and cardiovascular diseases.1,5,6,10–13 However, data on FABP4 in human atherosclerosis are scarce. The present study shows that FABP4 expression is high in unstable CPs and associates with the cardinal features of plaque vulnerability—lipid and macrophage accumulation, ulcerations, IPHs—and with ER stress and increased apoptosis. Our results suggest that a naturally occurring genetic low-expression variant of FABP4 associates not only with lower circulating serum total-C levels and reduced FABP4 expression and apoptosis within the carotid atheroma but also with lower risk for developing large artery atherosclerosis in metabolically strained obese individuals on a population level. In line, a greater proportion of patients with clinically asymptomatic advanced carotid disease were carriers of the low-expression variant when compared with patients with symptomatic disease. Furthermore, CC homozygotes, with the largest reduction in serum total-C levels, also showed an 8-fold reduced odds for MI and a trend toward lower frequency of all ischemic cardiovascular events. Therefore, FABP4 rs77878271 may represent a novel variant affecting both serum total-C levels and cardiovascular risk and suggests that genetically reduced FABP4 expression within the atherosclerotic plaque may modulate ER stress signaling and attenuate apoptosis, lipid burden, and inflammation with a combined effect of promoting atherosclerotic plaque stability.
Circulating total and LDL-C levels are heritable risk factors for cardiovascular diseases, and as such subject for active therapeutic intervention.34 We report here a novel association between the low-expression variant of FABP4 and reduced serum total-C levels, with the most prominent effect in CC homozygotes (Figure 1A). This is in line with a previous result suggesting a trend toward lower apoB levels in the low-expression variant carriers.22 The reduction in serum total-C and apoB levels was most prominent, ≈−16% and -20%, respectively, in the highest BMI quartile among the CC homozygotes (Figure 1E). These results suggest that the reduction in serum total-C might, in part, be mediated via other apoB-containing particles than LDL. In aP2−/− mice, on either normal chow or on high fat diet, circulating cholesterol levels remain unaffected.1 However, when crossed to a leptin-deficient background (ob/ob-aP2−/−), a genetic model of obesity, the mice show a significant reduction in circulating cholesterol and triglyceride levels.13 The enhanced reduction in serum total-C and apoB levels in the highest BMI quartile of CC homozygotes suggests that body weight and the cholesterol-lowering effect might be mechanistically linked also in man. One such mechanistic option is that the adipose tissue–specific reduction of FABP4 expression seen in the low-expression variant carriers22 mediates analogous enhancement of adipocyte de novo lipogenesis as reported in the lipid chaperone-deficient mice with reduced cholesterol levels and highly beneficial metabolic phenotype.4,35 At present, no evidence exists linking reduced FABP4 expression in white adipose tissue to enhancement of de novo lipogenesis and improved insulin sensitivity in man. However, recent evidence indicates that activated de novo lipogenesis as an independent mechanism predicts metabolic health in humans.36
In the Health 2000 population cohort, we discovered that the low-expression variant of FABP4 was associated with reduced carotid IMT among obese (BMI≥30) carriers (Table 2). Higher carotid IMT is a marker of subclinical atherosclerosis that directly associates with increased risk for MI and stroke.37,38 In line with reduced IMT, the obese low-expression variant carriers also demonstrate 3× less atherosclerotic CPs (Table 2). Importantly, these atheroprotective effects of the low-expression variant manifest during obesity, a metabolically stressed state. Consistently in aP2-deficient mice, obesity has been shown to trigger the emergence of a favorable metabolic phenotype characterized with improved lipid metabolism, peripheral insulin action, and preserved pancreatic β-cell function resulting in protection against obesity-induced insulin resistance and type 2 diabetes mellitus.1,13 Our results indicate a previously unrecognized role for the low-expression variant of FABP4 in reducing pathological arterial remodeling and retarding atheroma progression in metabolically challenged high-risk individuals. However, additional studies in larger cohorts are needed to confirm the association between reduced carotid IMT and the low-expression variant of FABP4 in obese individuals.
The CC homozygotes showed a low rate of cardiovascular events at baseline and there was only 1 new case of stroke and no new cases of MI or major coronary events during 10-year follow-up (Table 1). However, the small homozygosity frequency (0.4%) limited statistical power in the end point analyses of the population cohort, and the cardioprotective effect of this genotype was borderline significant (P=0.073; Table 1). When pooling all prevalent cases in the Health 2000 and the Corogene data sets, CC homozygotes showed an 8-fold reduced odds for MI (P=0.019; odds ratio, 0.12; 95% CI, 0.003–0.801). This finding is substantiated by our novel observation of reduced serum total-C levels, one of the most important risk factors of MI, in the low-expression variant carriers with the most prominent cholesterol-lowering effect coinciding with the 8-fold lower odds for MI. This is also in line with the reported atheroprotective effect of the low-expression variant on coronary heart disease22 and the atheroprotective role of FABP4 deficiency in mice.5,10,11 The rs77878271 variant has not been included in the recent large meta-analyses of genome-wide association studies analyzing genetic variants associated with MI, stroke, or plasma lipid traits, and hence explains why it has remained undetected.34,39,40
FABP4 expression in the CPs was detected in the same histological areas together with successively phosphoactivated ER stress markers and positively associated with apoptosis markers, aCASP3 and TUNEL reactivity (Figure 5). Consistently, the carriers of the low-expression variant of FABP4 had a 2.7-fold reduction in the relative density of aCASP3 immunoreactive cells and showed a trend toward lower TUNEL reactivity in their CPs (Figure 5D and 5E). The genetic deficiency or chemical inhibition of FABP4 has been shown to reduce lipotoxic ER stress and macrophage apoptosis in atherosclerotic lesions of mice.5 In humans, UPR-mediated macrophage apoptosis in advanced atherosclerotic lesions has been postulated to require several simultaneous noxious ER stress signals to trigger cell death.16,33 Perhaps, by reducing FABP4 expression and consequently FABP4-mediated lipotoxic ER stress, the atheroma cells in the low-expression variant carriers are able to evade apoptosis in situations that otherwise would lead to cell death. It is intriguing to speculate that similar enhancement of de novo lipogenesis with generation of protective bioactive lipids, such as C16:1n7-palmitoleate, that is afforded by FABP4 deficiency in mice could also promote macrophage survival in atherosclerotic lesions of the low-expression variant carriers.5 These results suggest that the low-expression variant is not only associated with reduced FABP4 transcription but also could confer an effect on cell viability in the atherosclerotic lesion.
In conclusion, we report here novel data on multiple levels of evidence suggesting an important role for FABP4 in pathophysiological cascades of human atherosclerotic disease. This evidence suggests that a naturally occurring genetic low-expression variant is atheroprotective and associates with reduced serum total-C levels and FABP4 expression in atherosclerotic lesions thus promoting plaque stability and reducing the risk of cardiovascular events. Our data on human atherosclerotic plaques support the view that genetically reduced FABP4 expression leads to an alleviation of ER stress as proposed by previous studies in deficient animal models. In mice, an orally active small-molecule inhibitor of aP2 has been demonstrated not only to prevent atherosclerosis development but also to retard already formed vascular lesions.6 Thus, pharmacological inhibition of FABP4 may be an effective strategy to manage atherosclerosis progression and to prevent its serious thromboembolic complications, stroke, and premature cardiovascular death.
Nancy Lim and Taru Puhakka are thanked for skilful technical assistance.
Sources of Funding
This study was funded by Helsinki University Central Hospital research grants, the Academy of Finland and the Sigrid Jusélius, Lundbeck, Aarne and Aili Turunen, Finnish Angiology, The Ida Montin, The Finnish Medical, Finnish Brain, Maire Taponen and Paavo Nurmi Foundations. Wihuri Research Institute is maintained by the Jenny and Antti Wihuri Foundation.
The Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.113.000499/-/DC1.
- Received December 5, 2012.
- Accepted June 25, 2014.
- © 2014 American Heart Association, Inc.
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Fatty acid–binding protein 4 (FABP4 or aP2 in mice) has been identified as a key regulator of core aspects of cardiometabolic disorders, including lipotoxicity-mediated endoplasmic reticulum stress in macrophages. However, the significance of FABP4 in human atherosclerosis remains unclear. Here, we demonstrate that FABP4 is overexpressed in atherosclerotic carotid plaques causing thromboembolic strokes. We show that a naturally occurring low-expression variant of FABP4 (rs77878271) is associated with reduced FABP4 transcription within the atherosclerotic plaque and that it restricts the development of large artery atherosclerosis during obesity and reduces lesion vulnerability and associated atherotrombotic complications on a population level. We also found that the low-expression variant was associated with reduced total cholesterol levels with the largest reduction in variant allele homozygotes. Consistently, the variant allele homozygotes showed 8-fold lower odds for MI. The cholesterol-lowering effect in the variant allele homozygotes was enhanced by almost 3-fold in the highest body mass index quartile. These data reveal FABP4 rs77878271 as a novel variant affecting serum total cholesterol levels and cardiovascular risk. This is the first evidence connecting genetically reduced expression of the main adipocyte lipid chaperone, FABP4, to cholesterol metabolism in man. This raises the notion that downregulation of FABP4 could mediate analogous enhancement of adipocyte de novo lipogenesis in man as reported in murine combined lipid chaperone deficiency, with the largest effect in obese individuals. A therapeutic regimen reducing FABP4 expression within the atherosclerotic plaque may promote lesion stability through modulation of endoplasmic reticulum stress signaling, attenuation of apoptosis, lipid burden, and inflammation.