Carotid artery atherosclerosis in hypertensive patients with a functional LDL receptor-related protein 6 gene variant

Nutrition, Metabolism & Cardiovascular Diseases (2011) 21, 150e156 available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/nmcd

Carotid artery atherosclerosis in hypertensive patients with a functional LDL receptor-related protein 6 gene variant R. Sarzani a,*, F. Salvi a, M. Bordicchia a, F. Guerra a, I. Battistoni a, G. Pagliariccio b, L. Carbonari b, P. Dessı`-Fulgheri a, A. Rappelli a a

Department of Internal Medicine, ‘‘Hypertension Excellence Centre’’ of the European Society of Hypertension, University ‘‘Politecnica delle Marche’’, Ancona, Italy b Department of Vascular Surgery, University ‘‘Politecnica delle Marche’’, Ancona, Italy Received 9 February 2009; received in revised form 17 June 2009; accepted 4 August 2009

KEYWORDS Hypertension; Carotid atherosclerosis; Genetics; Lipoprotein receptorrelated protein 6; Wnt/b-catenin

Abstract Background and aims: Rare (611C) and common (1062V) variants of the LowDensity Lipoprotein Receptor-Related Protein 6 (LRP6) display reduced activation of Wnt/ß-catenin signaling. The rare gene variant was associated with hypertension, metabolic abnormalities, and early coronary artery disease. We investigated whether the common 1062V LRP6 variant was related to carotid artery atherosclerosis (CAA) in hypertensive patients. Methods and results: Retrospective study of 334 hypertensive patients (<65 years old) who underwent carotid artery ultrasonography. Hypertension, type 2 diabetes, dyslipidemia, glomerular filtration rate, and smoking habit were evaluated. CAA was defined by the presence of atherosclerotic plaques (focal intimaemedia thickness 1.3 mm). Logistic regression models were used to estimate the independent effect of 1062V allele. The relationship between LRP6 genotypes and LRP6 gene expression in carotid plaques was also investigated. No difference was observed between genotypes in clinical variables except for a slightly higher fasting glucose in 1062V carriers. The 1062V LRP6 variant was an independent risk factor for CAA in both unadjusted (OR 2.08, 95%CI 1.27e3.41, p Z 0.003) and adjusted models (OR 1.92, 95%CI 1.09e3.39, p Z 0.02). LRP6 was expressed in carotid atherosclerotic plaques at significantly lower levels (p Z 0.015) in 1062V carriers.

* Correspondence to: Department of Internal Medicine, University of Ancona ‘‘Politecnica delle Marche’’, University Hospital ‘‘Ospedali Riuniti’’, 60131 Ancona, Italy. Tel.: þ39 71 889499; fax: þ39 71 889232. E-mail address: [email protected] (R. Sarzani). 0939-4753/$ - see front matter ª 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.numecd.2009.08.004

LRP6 alleles and carotid atherosclerosis

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Conclusion: Beside the role of established risk factors, 1062V variant of LRP6 and CAA are strongly associated in hypertensive patients, making LRP6 a novel relevant candidate gene for atherosclerosis in the presence of hypertension. ª 2009 Elsevier B.V. All rights reserved.

Introduction

Methods

Carotid artery atherosclerosis (CAA) is one of the main underlying causes of ischemic stroke [1]. Epidemiological studies have defined both established (age, male gender, hypertension, dyslipidemia, diabetes, smoking habits) and emerging (e.g. chronic kidney damage) risk factors for CAA [2]. Genetic predisposition plays a key role too: carotid artery intimaemedia thickness and plaques are highly heritable [3,4] and several candidate genes have been investigated [5e8]. Despite that, genetic factors differently influence plaque growth in patients with similar atherogenic phenotype (e.g. hypertension and dyslipidemia). A major gene for atherosclerosis acting independently of known risk factors has not been identified yet. Recently, the Wnt/b-catenin signaling pathway has been involved in vascular development and remodeling [9,10]. This pathway regulates many steps involved in cell growth, migration, differentiation, and fate. Wnts are a large family of secreted lipid-modified proteins that bind to sevenmembrane-spanning receptors termed ‘‘Frizzled’’. Wnt binding allows b-catenin translocation to the nucleus [11], where a family of transcription factors (T-cell factors/ lymphoid-enhancing factors: TCF/LEF) is activated and is involved in the regulation of glucose metabolism and insulin secretion. Low-Density Lipoprotein (LDL) Receptor-Related Protein 6 is a coreceptor in this ‘‘canonical’’ Wnt signaling pathway involved also in the regulation of glucose and lipid metabolism. A very rare mutation (R611C) in the LRP6 gene impairs b-catenin signaling and has been recently suggested to be responsible for hypertension, type 2 diabetes, high LDL cholesterol and triglycerides, and early coronary artery disease [12]. A more common LRP6 variant (I1062V; rs2302685) similarly impairs b-catenin signaling and has been associated with definite or probable late-onset Alzheimer’s disease [13]. Both mutations are in Epidermal Growth Factor (EGF)-like domains and have essentially identical biochemical profile. LRP6 gene is expressed in vascular smooth muscle cells (VSMC) and in rat carotid arteries [9,14], and its functional variants appear to be involved in degenerative disorders and atherosclerosis, similarly to what happens for other LRP family members (i.e. LRP1) [15,16]. Thus, the aim of the present study was to investigate whether the common functional LRP6 1062V variant was related to CAA in hypertensive patients. Indeed, it is interesting to note that the ‘‘index case’’ of the rare variant with homozygous LRP6 mutation (C611C) presented with myocardial infarction at age 48 but had progressive atherosclerosis of internal carotid arteries and died from a stroke at age 72 [12]. Therefore, we also studied the LRP6 gene expression in human carotid atherosclerotic plaques analyzing the relationship between LRP6 variants and LRP6 expression levels in vivo.

Study population This retrospective study involved 334 consecutive hypertensive patients (<65 years old) referred to our Hypertension Centre, who underwent carotid artery Doppler ultrasonography as part of target-organ damage assessment, following indications of the 2003 ESH-ESC Hypertension Guidelines [17]. The study was approved by the local Ethics Committee and each participant gave a written informed consent. Hypertension was defined as systolic blood pressure (SBP) 140 mmHg and/or diastolic blood pressure (DBP) 90 mmHg, or current therapy with anti-hypertensive drugs. Blood pressure (BP) was measured by a physician three times in sitting position, using a standard mercury sphygmomanometer with appropriate cuff size. Pulse pressure was calculated as systolic BP minus diastolic BP (average of three measurements). Diabetes was defined as fasting glucose 126 mg/dl, or a positive (fasting glucose 200 mg/dl) 2 h oral glucose tolerance test (performed when fasting glucose was 100 and <126 mg/dl), or therapy with anti-diabetic drugs or insulin. Dyslipidemia was defined as LDL cholesterol 160 mg/dl, or HDL cholesterol <40 mg/dl in males (<50 mg/dl in females), or triglycerides 150 mg/dl, or therapy with statins or fibrates. Fasting glucose, creatinine, apolipoproteins and lipid profile were measured with commercial kits used in the certified (ISO 9001:2000) University Hospital Central Laboratory; glomerular filtration rate (GFR) was calculated using the Modification of Diet in Renal Disease (MDRD) Study equation [18]. Apolipoprotein (Apo) A1 and Apo B-100 levels were measured in a subgroup of 235 patients. Smoking habit was evaluated as dichotomous variable (yes/never).

Carotid ultrasound examination Carotid arteries were assessed by duplex scan ultrasonography (ATL HDI 5000, Philips) and the results of the ultrasound examinations were reported in a clinical electronic database in a descriptive manner (i.e. normal, thickened, plaque). When an atherosclerotic plaque was present, it was quantified as percentage of lumen reduction and classified following Taylor and Strandness [19]. In our ultrasound laboratory, the intimaemedia thickness (IMT) cut-offs used to define thickening or plaque were 0.9 mm [17] and 1.3 mm [20], respectively, independently of the site (common, bifurcation, or internal carotid, bilaterally). The IMT values for each patient were not available because of the clinically oriented records. Reliability of ultrasonic measurements in our Centre was previously reported [7].

152

LRP6 I1062V genotyping and expression analysis Genotyping was carried out using a TaqMan SNP Genotyping Assay (C__15757990_10, Applied Biosystems, Darmstadt, Germany). A standard PCR was performed in a 25 ml volume containing 10 ng genomic DNA, 12.5 ml 2X TaqMan Universal PCR Master Mix (Applied Biosystem) and 1.25 ml 20X SNP Genotyping Assay Mix. Negative quality controls were included in all analyses and 10% of samples was duplicated and scored blind. Expression of LRP6 gene in human carotid plaques was investigated by using Real Time PCR. Human carotid plaque samples were obtained from further 34 older hypertensive patients (29 males, mean age 72.4  6.6 years; BMI 26.8  6.2 kg/m2; SBP 145.2  14.6 mmHg; DBP 79.6  11.5 mmHg) undergoing carotid endarterectomy at the University Hospital of Ancona (Italy) for asymptomatic carotid artery stenosis 70%, as confirmed by CT, MR or angiography. RNA was isolated from carotid plaques (frozen in liquid nitrogen after surgery) by using PureLink Micro-toMidi (Invitrogen) according to the manufacturer. Total RNA was converted to single stranded cDNA using High-Capacity cDNA Archive Kit according to the manufacturer (Applied Biosystems). Real Time PCR was performed in triplicate by using TaqMan Gene Expression Assay (Hs00999791_m1, Applied Biosystem). Differences in starting total RNA and different efficiency of cDNA synthesis were normalized using 18S rRNA expression as housekeeping gene.

Statistical analysis HardyeWeinberg equilibrium (HWE) was tested with c2 test. Analysis of variance (adjusting for age, gender, and BMI) was used to analyze differences in clinical variables between genotypes. Two different definitions of CAA ‘‘cases’’ and ‘‘controls’’ were used: in the first one, patients with an atherosclerotic plaque were considered as ‘‘cases’’ and patients without plaques (normal or thickened) as ‘‘controls’’; in the second one, patients with an atherosclerotic plaque producing more than 15% lumen reduction (used only as an index of larger plaque despite of the absence of significant hemodynamic effect) were classified as ‘‘cases’’ whereas patients without plaques or with plaques producing less than 15% carotid artery stenosis were ‘‘controls’’ [21]. The unadjusted odds ratio for CAA in subjects carrying 1062V variant was calculated. Logistic regression analysis was then used to confirm the independent effect of this variant on CAA. Variables entered in the logistic regression model (age, sex, smoking habit, diabetes, dyslipidemia, and pulse pressure) were selected by univariate analysis using a large (n Z 635) cohort of hypertensive patients including the genotyped population. The clinical characteristics of the non-genotyped (DNA not available) subjects did not differ from those of the genotyped subset. Categorical variables (i.e. diabetes, dyslipidemia) have been used instead of continuous values (i.e. fasting glucose, cholesterol levels) to avoid influence of therapy on laboratory data. Difference between genotypes in LRP6 gene expression levels in carotid atherosclerotic plaques of further 34 older patients undergoing endarterectomy was tested using the

R. Sarzani et al. Student’s t-test using the I1062I homozygous group as the calibrator. The cycle number (Ct) for 18S was subtracted from the Ct of the gene of interest (in this case LRP6), resulting in the difference in Ct, that is, DCt. The average DCt was then calculated for the calibrator group and this value was subtracted from every DCt value in all groups, resulting in a DDCt value for all samples. The DDCt value was then entered into the equation 2DDCt, which resulted in a fold induction value. All groups were compared with the calibrator group (which has a value of 1). Statistical analysis was performed with SPSS 13.0 (SPSS Inc. Chicago, IL, USA). A level of p < 0.05 was considered as significant.

Results Clinical characteristics of the population are shown in Table 1. Genotype distribution respected the HWE (p Z 0.86). Because of the low number of homozygous for 1062V allele (n Z 8), subjects were grouped in carriers (n Z 92) and non-carriers (n Z 242). No difference was found between carriers and non-carriers regarding clinical variables assessed except for an increased fasting glucose in LRP6 1062V subjects (Table 1) considering only patients free from therapy for diabetes. Atherosclerotic plaques were present in 167 patients (50%). As shown in Table 2, the 1062V LRP6 variant was an independent risk factor in both unadjusted (OR 2.08, 95%CI 1.27e3.41, p Z 0.005) and adjusted (age, gender, pulse pressure, dyslipidemia, diabetes, and smoking) models (OR 1.92, 95%CI 1.09e3.39, p Z 0.02). Moreover, using the larger cut-off to define CAA [atherosclerotic plaques with >15% lumen reduction [21]] 88 patients (26.3%) had CAA and the 1062V allele confirmed its strong role in both unadjusted (OR 2.7, 95%CI 1.61e4.54, p < 0.0001) and adjusted (OR 2.48, 95%CI 1.4e4.4, p Z 0.002) models (Table 3) suggesting a role in plaque growth. Despite the limitation due to the low number of V1062V homozygous subjects, statistical analysis considering the three genotypes revealed a dose-dependent effect of the 1062V allele on CAA: in unadjusted analysis, 1062V homozygous hypertensive patients had an OR of 8.54 (95%CI 1.04e70.49, p Z 0.046) for atherosclerotic plaques and an OR of 26.88 (95%CI 3.23e223.55, p Z 0.002) for carotid stenosis >15% of the lumen (Supplementary data). LRP6 expression in carotid atherosclerotic plaques of further 34 hypertensive patients with hemodynamically significant carotid stenosis (>70%) was evaluated. LRP6 transcripts were present in carotid artery plaques (Fig. 1A), suggesting that this coreceptor for Wnt/b-catenin signaling may be functional in human atherosclerotic plaques in vivo. Genotyping of these 34 patients revealed 24 homozygous I1062I and 10 heterozygous I1062V, corresponding to 29.4% prevalence of the 1062V variant. Significantly lower LRP6 expression levels (p Z 0.015) were present in 1062V carriers compared with I1062I homozygous subjects (Fig. 1B).

Discussion Classic and emergent risk factors are involved in the development of CAA as much as genetic predisposition. Heritability was estimated ranging between 20% and 40% of

LRP6 alleles and carotid atherosclerosis Table 1

153

Clinical characteristics of the hypertensive population.

Characteristic

Whole population (n Z 334)

LRP6 I1062I (n Z 242)

LRP6 V1062 carriers (n Z 92)

p

Age (years) Males/Females Smokers/Never smoked BMI (kg/m2) SBP (mmHg) DBP (mmHg) PP (mmHg) Anti-hypertensive drugs (yes/no) Creatinine (mg/dl) GFR (ml/min/1.73 m2) Diabetes (yes/no) Hypoglycemic drugs (yes/no) Fasting glucose (mg/dl)a Dyslipidemia (yes/no) Lipid-lowering drugs (yes/no) Total cholesterola (mg/dl) HDL cholesterola (mg/dl) Triglyceridesa (mg/dl) LDL cholesterola (mg/dl) ApoA1a (mg/dl) ApoBa (mg/dl) ApoB/A1 ratioa

52.7  9.2 212/122 157/177 31.9  5.6 150.3  21.0 92.7  13.0 57.8  16.2 282/52 1.06  0.30 74.0  16.0 75/259 15/319 100.1  25.2 248/86 46/288 208.3  42.2 44.9  12.8 159.2  99.6 132.5  38.7 158.8  45.5 116.1  48.7 0.76  0.36

52.2  9.5 149/93 107/135 32.2 (0.4) 149.7 (1.4) 92.4 (0.8) 57.6 (1.1) 203/39 1.03 (0.02) 73.7 (1.1) 50/192 10/232 97.6 (1.7) 180/62 31/211 210.2 (2.9) 46.8 (0.9) 149.5 (6.6) 134.3 (2.7) 164.4 (5.0) 116.0 (5.3) 0.72 (0.04)

54.0  8.4 63/29 50/42 32.0 (0.6) 151.4 (2.4) 92.7 (1.4) 58.6 (1.8) 79/13 1.03 (0.03) 74.4 (1.7) 25/67 5/87 104.5 (3.0) 68/24 15/77 204.7 (5.1) 44.0 (1.5) 149.0 (11.6) 132.4 (4.6) 156.3 (8.3) 114.6 (8.6) 0.77 (0.06)

0.13 0.24b 0.10b 0.75 0.53 0.82 0.61 0.66b 0.82 0.72 0.24b 0.57b 0.04 0.93b 0.41b 0.34 0.12 0.97 0.73 0.43 0.90 0.49

Results of analysis of variance (ANOVA). Data are mean  SD or absolute number. Data adjusted for age, gender, and BMI are presented as adjusted mean (SE); GFR is adjusted only for BMI. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; PP, pulse pressure; GFR, glomerular filtration rate as calculated by MDRD formula; HDL, high-density lipoproteins; LDL, low-density lipoproteins; ApoA1, apolipoprotein A-1; ApoB, apolipoprotein B-100. a Patients on active treatment with statins or fibrates (hypoglycemic drugs for fasting glucose) were excluded to evaluate the net effect of LRP6 alleles using data free from the drug effects. Data did not substantially change when including patients taking lipidlowering medications. b Results of c2 test.

IMT variability [6], depending on the population studied. Plaques as carotid atherosclerosis phenotype were not studied as frequently as carotid IMT despite that their heritability was estimated at 23%e28% [4]. Several genetic variants have been examined in relation to carotid atherosclerosis, mostly by association studies [6]. Disappointingly, a major gene for atherosclerosis has not been identified yet. A recently described rare mutation in LRP6 gene suggested a new common basis for traditional risk Table 2

factors leading to early coronary artery disease [12], similarly to what happens for polymorphisms of other LRP family members [15,16]. Thus, this study aimed at investigating the association between a common variant in LRP6 gene and CAA in hypertensive patients. Beside the role of established risk factors for atherosclerosis, our data in hypertensive patients indicated a strong genetic link between LRP6 1062V variant and CAA. The relatively young (below 65 years) patients studied are

Risk factors for CAA in the hypertensive patients genotyped for the I1062V polymorphism.

LRP6 1062I homozygous 1062V carriers Age Gender (male) Dyslipidemia Diabetes Smoking habit PP

No CAA

CAA

Unadjusted OR (95%CI)

p

Adjusted OR (95%CI)

p

133 (79.6) 34 (20.4)

109 (65.3) 58 (34.7)

2.08 (1.27e3.41)

0.005

1.92 (1.09e3.38)

0.02

1.13 (1.09e1.17) 1.96(1.14e3.35) 1.69(0.93e3.07) 0.91(0.50e1.66) 1.39(0.82e2.35) 1.01(1.00e1.03)

<0.0001 0.02 0.09 0.76 0.23 0.14

The results are Fisher’s Exact Test and logistic regression analysis. Nagelkerke R Square of the logistic regression model Z 0.33. PP, pulse pressure.

154 Table 3

R. Sarzani et al. Risk factors for CAA (carotid stenosis >15%) in the hypertensive patients genotyped for the I1062V polymorphism. No CAA CAA Unadjusted OR p (carotid stenosis Z <15%) (carotid stenosis >15%) (95%CI)

LRP6 1062I homozygous 192 (78) 1062V carriers 54 (22) Age Gender (male) Dyslipidemia Diabetes Smoking habit PP

50 (56.8) 38 (43.2)

Adjusted OR (95%CI)

2.7 (1.61e4.54) <0.0001 2.48 (1.40e4.39) 1.11 2.07 1.56 1.10 1.58 1.01

p

0.002

(1.07e1.16) <0.0001 (1.12e3.83) 0.02 (0.79e3.07) 0.20 (0.59e2.06) 0.77 (0.88e2.81) 0.12 (0.99e1.03) 0.22

The results are Fisher’s Exact Test and logistic regression analysis. Nagelkerke R Square of the logistic regression model Z 0.27. PP, pulse pressure.

likely to be a good model to investigate the effect of a single gene, and our results indeed supported a role for this single gene variant in CAA. The LRP6 expression in carotid plaques, the lower LRP6 expression levels in 1062V carriers, and the previously reported reduced activity of the 1062V variant [13] suggest local modulation of Wnt/bcatenin signaling resulting in reduced anti-apoptotic activity of this pathway [22] or impaired cellular LDL clearance [23]. LRP6 is expressed in VSMC [9] and the Wnt/b-catenin signaling pathway is involved in the pathogenesis of vascular disease and remodeling, regulating VSMC proliferation, survival, and apoptosis in the context of vascular injury [9,14]. Endothelial and VSMC show b-catenin accumulation in the cytoplasm or nucleus during vascular remodeling following disease state or acute damage, such as balloon injury [9]. This finding is a consequence of Wnt signaling that promotes cell cycle progression and inhibits apoptosis [9]. It is conceivable that the Wnt cascade results in an acute response of tissue repair and endothelial protection aimed to maintain homeostasis [24]. Therefore, a defective Wnt/LRP6/b-catenin signaling could be a cornerstone in developing atherosclerosis. Interestingly, the LRP6 1062V allele is a defective variant [13] that shows decreased ligand-independent and minimal decrease in ligand-dependent activation of b-catenin signaling. This biochemical profile is very similar to that of the 611C mutation responsible for hypertension, glucose and lipid dysmetabolism, and early coronary artery atherosclerosis [12]. In our hypertensive patients, the 1062V variant was associated with higher fasting glucose but not with lipid profile or diabetes prevalence (Table 1). Indeed, this same variant and other twelve single nucleotide polymorphisms of LRP6 have recently failed to associate with either susceptibility to type 2 diabetes or BMI, HOMA-indexes, and serum lipid concentrations [25]. Such data, together with ours, suggest that plaque growth in 1062V carriers may be partially independent of risk factors, in contrast with the high cardiovascular risk of the ‘‘index case’’ described by Mani et al [12] in which severe and early hypertension, diabetes and dyslipidemia were mainly responsible for early coronary and carotid artery disease. Indeed, a very recent paper has shown reduced membrane expression and impaired cellular LDL clearance in mice

carriers the 611C allele [23]. However, 1062V variant has been also associated with another ‘‘degenerative’’ disorder such as Alzheimer disease [13], supporting the possibility of pleiotropic effects in tissues where LRP6 is expressed. Accordingly, we found that LRP6 is expressed in atherosclerotic plaques and that 1062V carriers have reduced LRP6 expression. The similar LRP6 protein levels in cells transfected with the 1062V variant [13] might indicate that, in 1062V carriers, the cell composition of their plaques could be characterized by a lower number of viable cells expressing LRP6. This might be the result of an increased apoptosis of LRP6 1062V-harboring cells [9]. However, lack of concordance between gene and protein expression levels has been already described for LRP family members [26]. Moreover, because it is unlikely that a missense mutation variation in exon 14 could affect gene expression, it is possible that the functional variant we studied is linked to a still unknown functional variant in the promoter region, in introns or in introneexon junctions that may directly affect gene transcription (as happens for LRP1 gene) [16]. Some limitations of our study should be taken into account. First, genotyping was limited to 334 patients. Nonetheless, this population was a subset of a larger (n Z 635) cohort of hypertensive patients afferent to our Centre (data not shown) and did not differ from non-genotyped subjects. Moreover, the analysis shown in Table 3 has an 82% statistical power [27], although power of the analysis shown in Table 2 is lower than recommended (59%). Finally, despite the low number of 1062V homozygous subjects, we were able to show a dosedependent effect of the 1062V allele on CAA, although this result should be considered to be tentative rather convincing evidence for a role in CAA. Thus, the premises are striking, especially when considering the reduced function characteristic of this genetic variant. Second, we found association between 1062V variant and atherosclerotic plaques but we were unable to study association with IMT as a continuous trait because of the retrospective nature of the study and characteristics of the clinically oriented ultrasound reports. Nevertheless, IMT, plaque, and carotid stenosis appear to be biologically and genetically distinct entities [28], and the presence of plaque was found to be more predictive of cardiovascular events than IMT in hypertensive patients [29]. Third, due to the retrospective nature of the study, we were unable to consider current and former smokers separately in

LRP6 alleles and carotid atherosclerosis

A

155

B

4

3

2

* 1

0 I1062I

I1062V

Figure 1 A) Upper band (white arrow) represents the expression of 18S rRNA (187 bp), whereas the lower line (black arrow) is LRP6 expression (87 bp) in carotid plaque of four I1062I homozygous patients. Molecular weight markers are in the right column. B) LRP6 expression levels in carotid atherosclerotic plaques according to the presence of the 1062V variant. Data are mean  SD and are given as relative difference (and range) of the normalized gene expression results with the I1062I homozygous patients (white column; n Z 24) used as calibrator group set equal to 1; dotted column refers to I1062V heterozygous patients (n Z 10). *p Z 0.015.

the statistical analyses. Fourth, the study of LRP6 gene expression in carotid plaques is somewhat limited by the number of patients studied and the lack of immunohistochemistry to identify the cell-type expressing LRP6. Thus, LRP6 variants with impaired Wnt/b-catenin signaling appear to be involved in early coronary and carotid artery atherosclerosis, at least in hypertensive patients. This novel pathway in atherogenesis appears to ‘‘accelerate’’ plaque formation and growth. Lower LRP6 1062V-mediated Wnt/b-catenin signaling might result in altered function and growth of plaque-forming smooth muscle cells. The 1062V variant of LRP6 has the potential to become one of the most readily available and powerful genetic marker of accelerated atherosclerosis. Once our data will be confirmed, hypertensive patients carrying the 1062V LRP6 variant should undergo clinical investigation to detect subclinical atherosclerosis. Further studies are needed to confirm this association and to define the mechanisms involved in the plaque forming process, in the hope of discovering novel therapies to slow the progression of atherosclerosis.

Conflict of Interest The Authors declare no conflict of interest.

Sources of Funding This study was supported by research grants from the Ministry of University and the University Politecnica delle Marche, Ancona (Italy), without any role in the design, execution, analysis and interpretation of data, or writing of the paper.

Acknowledgments We are grateful to Prof. Flavia Carle, Department of Clinical Medicine and Applied Biotechnologies, University Politecnica delle Marche, Ancona (Italy), for the statistical advice in the power calculation.

Appendix Supplementary data Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.numecd.2009. 08.004

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