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. Author manuscript; available in PMC: 2019 Jun 12.
Published in final edited form as: Int J Stroke. 2015 Sep 1;10(8):1247–1252. doi: 10.1111/ijs.12623

Sirtuin/uncoupling protein gene variants and carotid plaque area and morphology

Chuanhui Dong 1,†, David Della-Morte 1,2,3,†, Digna Cabral 1, Liyong Wang 4, Susan H Blanton 4, Chaturvedi Seemant 1, Ralph L Sacco 1,4,5, Tatjana Rundek 1,5,*
PMCID: PMC6561468  NIHMSID: NIHMS1012020  PMID: 26332421

Abstract

Background

Sirtuins and uncoupling proteins have been implicated in cardiovascular diseases by controlling oxidative stress.

Aims

We sought to investigate the association of sirtuins and uncoupling proteins single nucleotide polymorphisms with total carotid plaque area and morphology measured by ultra-sonographic gray scale median.

Methods

We analyzed 1356 stroke-free subjects (60% women, mean age = 68 ± 9 years) from the Northern Manhattan Study. Multiple linear regression models were used to evaluate the association of 85 single nucleotide polymorphisms in 11 sirtuins/uncoupling protein genes with total plaque area and gray scale median after controlling for demographics, vascular risk factors (RFs), and population stratification. We investigated effect modifications of these relationship by gender and RFs and performed stratified analysis if the interaction effect had P < 0·005.

Results

Among individuals with present plaque (55%), the mean total plaque area was 20·3 ± 20·8 mm2 and gray scale median 90 ± 29. After adjustment, SIRT6 rs107251 was significantly associated with total plaque area (β = 0·30 per copy of T allele increase, Bonferroni-corrected P = 0·005). T allele carriers of rs1430583 in UCP1 showed a decreased gray scale median in women but not in men. The minor allele carriers of rs4980329 and rs12363280 in SIRT3 had higher gray scale median in men but not in women. Variants in UCP3 gene were significantly associated with higher mean gray scale median in individuals with dyslipidemia.

Conclusion

Our findings suggest that polymorphisms in SIRT6/ UCP1 genes may be important for increased carotid plaque burden and echodensity, but translation of these findings to an individual risk of cerebrovascular events needs further investigation. Significant associations of rs1430583 in women, rs12363280 in men, and rs1685354 in those with dyslipidemia also deserve further investigations.

Keywords: atherosclerosis, carotid total plaque area, genetics, gray scale median, mitochondrial uncoupling proteins, sirtuins

Introduction

Atherosclerosis is a multifactorial disorder and underlying cause of most ischemic strokes and cardiovascular diseases (CVD) (1). Subclinical markers of atherosclerosis including carotid plaque (CP) and carotid intima media thickness (IMT) have been widely used to assess the risk of future clinical atherosclerotic disease (1). Interestingly, these markers may be distinct biological phenotypes of atherosclerosis (2). Recent studies have demonstrated that variation in carotid atherosclerosis is largely unexplained by known vascular risk factors (3,4), suggesting that other unaccounted factors, both environmental and genetic, play an important role in the determination of atherosclerosis.

Oxidative stress is clearly involved in mechanisms of atherosclerosis leading to vascular diseases mainly by the enhancement of endothelial dysfunction, and reduction of nitric oxide bioactivity (5). Therefore, genes controlling balance between production and removal of reactive oxygen species (ROS) such as sirtuin (SIRTs) (6) and mitochondrial uncoupling proteins (UCPs) genes (7) have been proposed in atherosclerosis. These processes may be pivotal in the regulation of plaque morphology since ROS can increase the expression of cell surface receptors required for uptake of oxidized low-density lipoprotein (8). SIRTs control the genetic expression of UCPs by binding directly to their promoters. The up- or down-regulation and the enzymatic activity of SIRT/UCP proteins have been related to the degree of tolerance to brain ischemia (9). Therefore, modulating SIRTs and UCPs functions may offer a promising strategy for combating atherosclerosis and CVD.

Previously, we demonstrated a significant association between several single nucleotide polymorphisms (SNPs) in SIRT/UCP genes and CP presence and number (10), IMT (11), and carotid artery stiffness (STIFF) (12), as well as effect modification of these associations by vascular risk factors. In this study, we extend these investigations to plaque burden measured by total plaque area (TPA) and plaque morphology measured by an ultrasonographic index of plaque echodensity or gray scale median (GSM).

TPA has been suggested as the most useful subclinical CVD measure to evaluate the effects of anti-atherosclerotic treatments (13,14). GSM represents a novel marker of plaque morphology with great potential for clinical use (15,16). Low GSM values correspond to echolucent plaques with features that include high lipid content, and a thin fibrous cap, whereas high GSM index correlates to echogenic plaques of high fibrous content and calcification (17). In our previous study, we demonstrated that subjects with CP calcification had a significant increased risk of the combined vascular events (18). Based on our previous discovered findings (10,12) and SIRT/UCP antioxidant properties, we hypothesize the significant role of SIRT/UCP genes in atherosclerosis burden and plaque morphology. We also hypothesized that these associations may be gender specific. Several reports have shown less lipid rich plaques in women (19,20), while others have reported more calcified plaques in men (21). In addition, major vascular risk factors may be significant modifiers of these associations.

Materials and methods

Study population

A sample of 1356 stroke-free participants was derived from the Northern Manhattan Study (NOMAS). We have reported the detailed ascertainment scheme of the NOMAS previously (22). Briefly, NOMAS participants were eligible if they had never been diagnosed with a stroke, were at least 40 years of age, and resided for at least three-months in a household with a telephone in northern Manhattan. At enrollment, demographic characteristics and RFs were collected through standardized questionnaires and laboratory tests. Hypertension was defined as a systolic BP ≥ 140 mmHg, diastolic BP ≥ 90 mmHg, or a history of hyper-tension and anti-hypertension treatment; diabetes mellitus was defined as fasting blood glucose ≥126 mg/dl or use of insulin or hypoglycemic medications; dyslipidemia was defined as total cholesterol ≥240 mg/dl or a history of taking lipid-lowering medications. Smoking was dichotomized as ever or never smoking, physical activity as any leisure-time activity or none, and alcohol drinking as light to moderate (≥1 drink/month but <2 drinks/day), none, or other. Body mass index (kg/m2) was calculated based on the measured weight and height. All subjects provided written informed consent to participate, and the study was approved by the Institutional Review Boards of Columbia University and the University of Miami.

Assessment of CP GSM and TPA

High-resolution B-mode ultrasounds (GE LogIQ 700, GE Health-care, Milwaukee, WI, USA, 9- to 13-MHz linear-array transducer) were performed by trained and certified sonographers as previously detailed (23). Left and right carotid bifurcations and internal and common carotid arteries were examined for the presence of plaque. Plaque was defined as an area of focal wall thickening 50% greater than surrounding wall thickness confirmed by marking and comparing plaque thickness with the thickness of the surrounding wall during scanning by electronic calipers (3). The plaque boundaries were traced offline from the digitized images using an automatic edge detection system M’Ath (Imt, Inc., Paris, France). The sum of plaque areas in all carotid arteries from both sides of the neck was expressed as a total CP area (TPA) (3). After image normalization using linear scaling, GSM of an operator-selected blood region inside the vessel lumen was mapped to 0, and the brightest region of the adventitia was mapped to 255 using automated software in M’Ath (24). Both of these reference regions were approximately 0·4 mm2 in area and were selected on the first image of the image sequence. The reference GSM values calculated on the first frame were applied to that and all subsequent images and GSM was expressed for each plaque separately. The mean GSM of all plaques within an individual was calculated and used in the analyses.

Genotyping

We identified variants in SIRT and UCP genes available from a genome-wide screen performed on the AffyMetrix Genome-Wide Human SNP Array 6.0 chip (Affymetrix, Inc., Santa Clara, CA, USA). The arrays were scanned on the Gene Chip Scanner 3000 7G (Affymetrix, Inc.), and image data were analyzed using the Genotyping Console™ (Affymetrix, Inc.). Subjects were removed from further analysis if they had call rates below 95%, relatedness, gender discrepancies, or were outliers beyond 6 SD from the mean using EIGENSTRAT (25). SNPs with severe deviation from Hardy–Weinberg equilibrium (P < 1E−06) or a genotyping call rate less than 95% were also removed using PLINK1.05 (26). After quality control, a total of 85 UCP/SIRT SNPs in the 11 UCP/SIRT genes were included in the final analysis of this study (10).

Statistical analyses

To reduce potential bias due to population stratification, we first performed principal component analysis to examine population substructure using EIGENSTRAT and included the top three principle components (PCAs) as genomic control variables in the genetic association analysis. To control for potential confounders, we also included demographics (age, gender) and vascular factors (smoking, physical activity, moderate alcohol consumption, obesity, hypertension, diabetes mellitus, and hypercholesterolemia) in the model. For single SNP-based association analyses, we examined the additive genetic effects of the UCP/SIRT variants on TPA and GSM using linear regression models, after adjusting for the demographic characteristics, vascular factors, and the top three PCAs. Given a total of 16 LD blocks were identified and 14 SNPs were not located in any LD block, Bonferroni corrections were done to control for multiple testing based on an effective number of 30 (10,27). Alternatively, multiple testing corrections were conducted based on gene set tests with 5000 permutations (corrected P for gene set test = empirical P × 11). To explore the effect modification of modifiable vascular factors on the associations between genetic variants and mean TPA and GSM, we examined SNP-by-gender and SNP-by-vascular factor interactions and performed stratified analyses if the interaction terms had P < 0·005.All the analyses were performed using plink1.05 and sas9.3 (SAS Institute Inc., Cary, NC, USA).

Results

Among 1356 subjects (mean age: 68 ± 9), 60% were women, 69% Hispanic, 19% Black, and 11% White. All subjects were stroke free and did not report any previous cardiovascular clinical events, including myocardial infarct and/or transient ischemic attack. Overall, 19% had diabetes, 52% were smokers (17% current smokers and 35% former smokers), 30% had dyslipidemia, and 71% hypertension (Table 1). Among those with plaque (55%), the mean TPA was 20·3 ± 20·8 mm2 (interquartile range, 7·0–25·6) and mean GSM 89·5 ± 28·5 (interquartile range, 67·0–109·0).

Table 1.

Characteristics of participants by plaque size (total plaque area) and morphology (gray scale median)

N % TPA, mm2 GSM
Mean SD Mean SD
All 1356 100 11 18 49 49
Age
 <70 744 55 7 15 40 48
 70 + 612 45 16–1 21 62 47
Gender
 Male 540 40 14 22 52 49
 Female 816 60 9 16 48 49
Race/Ethnicity
 White 153 11 14 17 57 47
 Black 255 19 14 22 57 50
 Hispanic 930 69 10 18 46 49
 Other 18 1 9 14 44 50
Ever smoking
 No 655 48 9 16 43 48
 Yes 701 51 13 20 56 50
Physical activity
 No 625 46 11 18 48 49
 Yes 731 53 11 19 50 49
Moderate drinking
 No 821 60 11 18 49 49
 Yes 535 39 11 18 50 49
Obesity
 No 946 70 12 19 52 50
 Yes 410 30 10 16 44 46
Diabetes
 No 1105 81 10 17 47 49
 Yes 251 18 15 23 59 48
Hypertension
 No 396 29 8 13 42 48
 Yes 960 71 13 20 53 49
Dyslipidemia
 No 956 70 11 18 47 49
 Yes 400 29 13 19 55 49
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Table 2 reports the SNPs associated with TPA or GSM with a nominal P < 0·05 after adjustment for demographics, vascular factors, and the top three PCAs. T allele carriers of rs107251 in SIRT6 were associated with higher mean GSM (β = 8·66, 95% CI = 2·99–14·63, P = 0·003) and larger TPA (β = 0·30, 95% CI = 0·14–0·45, P = 0·0002). After correction for multiple testing based on effective test number, rs107251 remained significantly associated with TPA (Bonferroni corrected P = 0·005). When the correction for multiple testing through permutation was performed, gene set analysis showed that SIRT6 gene was significantly associated with GSM (permution corrected P = 0·04) and TPA (permution corrected P = 0·002). In addition, C allele carriers of rs2841503 in SIRT5 tended to have higher mean GSM and larger TPA, and C allele carriers of rs5977233 in UCP5 appeared to have higher mean GSM, but none of these associations remained significant after correction for multiple testing.

Table 2.

SNPs associated with carotid plaque size (area, TPA) and morphology (GSM)

Gene Chr SNP BP Minor allele MAF Phenotype Beta (95% CI)* Nominal P Bonferroni-corrected P
SIRT5 6 rs2841503 13548834 C 0–08 TPA 0·20 (0·02, 0·38) 0–0322 0·625
GSM 7·10 (0·34, 13·87) 0–0399 0·705
SIRT6 19 rs107251 4176085 T 0–11 TPA 0·30 (0·14, 0·45) 0–00017 0·005
GSM 8·81 (2·99, 14·63) 0–0031 0·088
UCP5 X rs5977233 129454635 C 0–29 GSM 4·59 (0·23, 8·95) 0–0393 0·699
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*

Adjusted for age, gender, smoking, physical activity, moderate alcohol consumption, obesity, hypertension, diabetes mellitus, hypercholesterolemia, and the top three PCAs. The value reported in bold passed the Bonferroni’s multiple testing corrections adjustment.

Table 3 shows the interactions between SNPs and gender and vascular factors with a nominal P < 0·005 and the genetic effects stratified by gender and the status of the specific vascular risk factors. SNP-by-gender interaction was found for three potential regulatory SNPs, one in UCP1 (rs1430583), and two in SIRT3 gene (rs4980329 and rs12363280) in the association with mean GSM. Specifically, T allele carriers of rs1430583 in UCP1 showed a decreased GSM in women (β = −8·87, P = 0·002) but not in men (P = 0·25), while the minor allele carriers of rs4980329 (β = 12·53, P = 0·005) or rs12363280 (β = 11·62, P = 0·01) in SIRT3 had higher GSM in men but not in women (P for interaction ≤ 0·0031). For the interactions between SNP and vascular risk factors, the strongest interaction was found between SNP rs1685354 in UCP3 gene and hypercholesterolemia. Specifically, A allele of rs2734827 and G allele of rs1685354 in UCP3 were significantly associated with higher mean GSM (β = 9·5, P = 0·004; β = 11·56, P = 0·001, respectively) in individuals with hypercholesterolemia, but not in those without hypercholesterolemia. Similarly, also, G allele in rs1685354 of UCP3 was associated with increased TPA (β = 0·27, P = 0·004) in individuals with hypercholesterolemia, but not in those without hypercholesterolemia (P for interaction = 0·0007).

Table 3.

SNPs showing interaction with gender and vascular factors with P < 0·005 in the association with carotid plaque size (area, TPA) and morphology (GSM)

Gene Chr SNP BP Minor allele Phenotype Stratified analysis P for interaction
Beta (95% CI)* Men P Beta (95% CI)* Women P
UCP1 4 rs1430583 141486984 T GSM 4·04 (−2·77, 10·85) 0·2455 −8·87 (−14·35, −3·39) 0·0016 0·0048
SIRT3 11 rs12363280 231980 G GSM 12·53 (3·88, 21·17) 0·0047 −3·84 (−11·34, 3·66) 0·3158 0·0031
rs4980329 232598 T GSM 11·62 (2·86, 20·38) 0·0096 −5·39 (−12·62, 1·83) 0·1439 0·0024
Hypercholesterolemia (+) Hypercholesterolemia (–)
UCP3 11 rs1685354 73713591 G TPA 0·27 (0·09, 0·46) 0·0043 −0·11 (−0·22, 0·01) 0·0693 0·0007
GSM 11·56 (4·67, 18·44) 0·0011 −2·89 (−7·28, 1·51) 0·1984 0·0009
rs2734827 73716277 A GSM 9·5 (3·01, 15·99) 0·0044 −2·04 (−6·38, 2·31) 0·3587 0·0034
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*

Adjusted for age, gender if applicable, smoking, physical activity, moderate alcohol consumption, obesity, hypertension, diabetes mellitus, hypercholesterolemia if applicable, and the top three PCAs. Bold are reported the values that are statistically significant after the multiple adjustment.

Discussion

In this study, we report a significant association of SIRT6 SNP rs107251 with total TPA and morphology (GSM) and several potential effect modifications by gender and hypercholesterolemia. Lower GSM was associated with UCP1 among women but not among men. Greater GSM was associated with SIRT3 in men but not in women. Greater GSM was also associated with UCP3 among women and men with dyslipidemia. Present results provide further evidence that genetic variants in sirtuin and UCP genes contribute to atherosclerosis and that the SIRT/UCP pathway may be an attractive target for the anti-atherosclerotic therapies.

Both TPA and GSM have been proposed as novel markers of plaque atherosclerosis and predictive of incident CVD and stroke (14,16). Plaque size (TPA) rather than plaque presence (yes/no) may be more useful measure in targeting and monitoring individuals for more aggressive anti-atherosclerotic treatments (14). The ultrasonographic GSM index is a global measure of plaque echodensity and therefore an indicator of plaque morphology. Plaque composition is based on the hyperechoic or hypoechoic ultrasound signal (28). Similar to TPA, GSM has been recently used to evaluate the effects of anti-atherosclerotic therapies (15).

In our previous study (10), we observed that carriers of the SIRT6 rs107251 had an increased risk for CP. In the present study with a larger sample size, we provide further evidence that SIRT6 SNPs are associated with increased TPA. SIRT6 is one of the seven sirtuins, which functions as an ADP-ribosylase and NAD+-dependent deacylase of both acetyl groups and long-chain fatty-acyl groups (29). SIRT6 is predominantly localized in the cellular nucleus and is involved in DNA repair and lifespan extension (29). The role of SIRT6 in carotid atherosclerosis can be related to its proinflammatory as well as anti-inflammatory function depending on the context and cell type involved (29). SIRT6 deficiency causes a specific increase in glucose uptake in multiple cell types (30), and absence in SIRT6 results in accumulation of triglycerides (TG) (31). A recent study has shown that SIRT6 pathway mediates the switch of vascular smooth muscle cells between a quiescent contractile phenotype and a synthetic phenotype in response to cyclic strain (32). Therefore, the pleiotropic enzymatic activities of SIRT6 in atherosclerosis may be regulated by different SIRT6 gene variants. In particular, rs107251, that we reported in the association with CP presence (10), has also been associated with ulcerative colitis in a meta-analysis of 6 GWAS (33), emphasizing its proinflammatory role. In addition, subjects carrying rs107251 minor allele homozygotes (TT) have been sown to have a shorter lifespan (five-year mean survival) in a cohort of 3763 participants after controlling for aging-related risk factors, suggesting that this SIRT6 variant may predispose individuals for premature age-related diseases (34). Our results show an association of this SIRT6 SNP with atherosclerotic phenotypes regardless of age, which deserves further investigation.

Another finding of our study is the marginal association of rs2841503 in SIRT5, and rs5977233 in UCP5 with increased TPA and increased GSM. In our previous studies, we found the association between different SIRT5 and UCP5 variants with plaque presence, STIFF, and carotid diameters, suggesting a potential important role of these genes in carotid atherosclerosis structure and function (10,12). SIRT5 is a mitochondrial sirtuin that is upregulated by caloric restriction and is involved in ROS production (6). The physiological role of UCP5 has not been yet defined, but it has been proposed as a modulator of ATP production (35).

We observed gender-specific effect of genetic variants in UCP1 resulting in lower GSM in women but not in men. We found a gender interaction between UCP1 SNPs and CP presence in our previous study (10). Our results confirm the role of UCP1 gene in carotid atherosclerotic and extend them to associations with plaque morphology. Calcified plaque are more prevalent in men than in women suggesting a gender-specific interaction in this process (21), with smoking being most important factor associated with calcified plaque in men (36). Gender-specific associations observed in current study could be explained, at least in part, by the sex hormone modulation of UCP1 expression, which have been already found to control body weight (37). In our study, women carrying specific UCP1 genetic variant have lower GSM. Even though our study population is comprised of elderly people, the different gender imprinting of sex steroid hormones on the effect of UCP1 in the mechanisms leading to the atherosclerosis during aging could have had an important effect. Our results may have several clinical implications. The American Heart Association and the European Stroke Organization guidelines suggest including age as well as gender into account when selecting patients with carotid stenosis for carotid revascularization (38). In a recent study conducted among individuals with unilateral asymptomatic internal carotid artery stenosis, different athero-sclerotic phenotypes including stenosis progression and irregular plaque surface have had a relevant gender-specific variability in the risk of ipsilateral stroke (39). Moreover, women with symptomatic high-grade carotid stenosis benefit from carotid surgery while asymptomatic women have less benefit (40). Taking these reports together with our results further emphasize the importance of understanding the gender-specific genetic and environ mental interactions in the development of atherosclerosis that will drive the development of targeted and gender-specific CVD preventive strategies.

The UCP1 rs1430583 was also associated with variation in diastolic and systolic diameters of carotid artery in our previous study (12), suggesting its role in the regulation of carotid geometry and remodeling. The regulation of carotid artery diameter changes and carotid blood flow hemodynamics is pivotal in the development of atherosclerotic plaque (41).

We observed gender-specific effect of variants in SIRT3 resulting in greater GSM in men but not in women. The mitochondrial SIRT3 deacetylates numerous proteins involved in mechanisms associated with atherosclerosis including fatty acid β-oxidation, amino acid metabolism, the electron transport chain, and anti-oxidant defenses (6). A recent study however has demonstrated that SIRT3 deletion affected neither plaque burden nor features of plaque vulnerability (i.e., fibrous cap thickness and necrotic core diameter) (42). However, in the setting of increased oxidative stress (e.g. smoking that is more prevalent in men), SIRT3 effect may be present and deleterious in men.

Finally, we found an interaction between UCP3 SNPs and dyslipidemia. Specifically, genetic variances in UCP3 were associated with greater GSM and TPA only in individuals with increased cholesterol. UCP3 rs1685354 has been associated with a significant decrease in visceral fat mass in a Dutch men study (43). Other UCP3 genetic variants have been indicated as modifiers of increased serum lipid levels and indices of abdominal obesity (44).

Our results need to be taken with caution because of several limitations. We included a relatively small convenience sample of SNP from our genome-wide association study (85 SNPs in the SIRT and UCP genes) (10). We have not provided an independent validation of our results. However, SNPs that we found associated with CP burden and morphology in the present study have been associated with vascular diseases and risk factors in several other studies (43,45,46).

Conclusions

Despite the limitations previously reported, the results from our study may be particularly important. Variances in SIRT/UCP genes associated with CP burden and morphology may have a pivotal role in atherosclerosis. Some of these processes may be more pronounced among women or may be more expressed in different metabolic conditions such as dyslipidemia. Exploration of the specific role of SIRT/UCP variants in vascular disease may help develop more specific SIRT activator compounds that can restore impaired molecular pathways, and thereby prevent and reduce atherosclerosis and CVD. In addition, the significant SIRT/UCP variances associated with TPA and GSM observed in this study were not found in our previous analyses with IMT (11) and STIFF (12). These ‘inconsistent’ findings support the hypothesis that CP, IMT, and STIFF are different phenotypes of atherosclerosis and under different genetic control. Further research is imperative to confirm these results.

Funding:

This research was supported by the NIH/NINDS K24-NS062737 grant, James & Esther King Biomedical Research Program (2KN01), and Evelyn F. McKnight Brain Institute.

Footnotes

Conflict of interest: None declared.

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