The Gene Polymorphism of LOX1 Predicts the Incidence of LVH in Patients with Essential Hypertension

Hypertension is a major etiological factor for left ventricular hypertrophy (LVH). Left ventricular hypertrophy (LVH) has been considered as an independent risk factor of cardiovascular events and death [1,2,3,4,5]. Epidemiological studies show that the prevalence of left ventricular hypertrophy (LVH) in Chinese patients with essential hypertension (EH) was about 25% to 35% [6]. A number of environmental factors, including blood pressure level, duration of hypertension, age, obesity, and smoking status have been shown to contribute to the incidence of LVH in human [7,8,9,10]. Epidemiological studies have also showed that the individual genetic background plays an important role in the incidence of LVH [11,12]. A variety of studies suggested the polymorphisms of candidate genes may be used as molecular markers to predict the LVH [13,14,15,16,17]. Unfortunately, the results of these studies are often inconsistent or even controversial.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is the major receptor of oxidized low-density lipoproteins (ox-LDL) which regulates growth of a variety of cells and is important in inflammation, atherosclerosis, oxidative stress, and tissue remodeling [18,19,20,21]. LOX-1 is expressed in various cells, including endothelial cells, macrophages, and chondrocytes, and its expression is enhanced by proinflammatory cytokines [22]. A recent study showed that LOX-1 is a key modulator of the development of angiotensin II-induced hypertension and subsequent cardiac remodeling [23]. LOX-1 deletion alters signals of myocardial remodeling immediately after ischemia-reperfusion [24]. LOX-1 also affects cardiac fibroblast growth and collagen secretion [25].

The LOX1 gene, also known as OLR1, is located on the chromosome 12p13.1-p12.3. The LOX1 protein is synthesized as a 40-kDa precursor protein and is composed of four domains: an extracellular lectin-like domain at the C-terminal, a connecting neck domain, a transmembrane domain, and an N-terminal cytoplasmic domain [26].

Three single nucleotide polymorphism (SNPs), namely, intron 4 (G→A), intron 5(T→G), and 3′ UTR (T→C) in the LOX1 gene, have been previously reported [27]. Hou et al. reported that the LOX1 polymorphism is closely related to the serum CRP levels and the occurrence of albuminuria in Chinese hypertensive patients [28,29], suggesting the potential application of the LOX1 polymorphism as a molecular marker for hypertension-related complications. However, the association between the LOX1 polymorphisms and LVH in hypertensive subjects has not yet been reported. Given the role of LOX1 in the cardiac remodeling [23,24,25] , we postulate that the LOX1 gene polymorphisms may affect the occurrence of LVH in hypertensive patients. In this hospital-based case-control study, we enrolled EH patients to test this hypothesis.

A total of 536 patients diagnosed with EH in our hospital were recruited between April 2007 and September 2011. According to the presence or absence of LVH, subjects were divided into the LVH+ (EH with LVH, n=143) and LVH- (EH without LVH, n=393) groups. In order to avoid the effects of drug therapy on LVH, patients were matched by the baseline antihypertensive therapy and disease duration. The baseline therapy includes the angiotension converting enzyme inhibitors (ACEI), calcium-channel blocker (CCB), Angiotension II Type 1 Receptor Blocker (AT1 RB), β receptor blocker, and diuretics. Those who with secondary arterial hypertension, congenital heart disease, dilated, hypertrophic or restrictive cardiomyopathy, valvular heart diseases, pulmonary hypertension, coronary heart disease (including Ischemic cardiomyopathy), severe heart failure, lipid metabolism disorders, acute and chronic liver disorders were excluded. All patients were provided with an informed written consent. The study protocol was approved by the ethics committee in our hospital.

The clinical variables including diabetes mellitus (DM), alcohol intake, smoking status, weight, height, body mass index (BMI), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were acquired from the medical charts. The levels of serum creatinine (sCr), total cholesterol (TC), total triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and blood glucose were acquired.

The measurement of High-sensitivity C-reactive protein (hs-CRP) was performed using a commercially available high-sensitivity assay (Roche Diagnostics). Serum sLOX-1 levels were measured by a commercially available enzyme-linked immunosorbent assay kit. The assay is sensitive to the sLOX-1 level of 2.4 pg/mL and above with a coefficient of variation <5 %.

Genomic DNA was isolated by using the Puregene Systems DNA purification kit (GENTRA). Genotyping for the LOX1 intron 4 G/A (14 bp from the 5′end of exon 5), intron 5 T/G (27 bp from the start of exon 6), and 3′UTR T/C (188 bp from the stop codon) polymorphisms was done as previously described [13].

Echocardiography was conducted for all subjects using a Hewlett-Packard imaging system (Sonos 2500 model, California). LV mass (LVM) was calculated at end-diastole using the formula: 0.8 × 1.04[(IVSd + LVIDD + PWTd) - LVIDD] + 0.6 (IVSd: interventricular septal thickness, PWTd: posterior wall thickness, LVIDD: LV end-diastolic internal dimension), which yields the values closely related (R = 0.90) to the necropsy LV weight. LVM was divided by height, 2.7, to obtain the LVMI (LVM index). LVH was defined as LVMI > 49.2 g/2.7 m for men and >46.7 g/2.7 m for women [16]. The echocardiography measurement was performed by two experienced echo doctors who were blind to the study protocols. The intra-observer correlation coefficiency for two investigators was 0.925 and 0.903, respectively (both P <0.001). The inter-observer correlation coefficiency of two interviewers was 0.885 (P <0.001).

The clinical and biochemical characteristics of all enrolled patients are shown in Table 1. The LVH+ and LVH- groups were similar in age, sex, smoking status, BMI, DBP, TG, TC, HDL, LDL and sCr (all P>0.05). The EH duration and therapy duration as well as the antihypertensive medication were also similar between the LVH+ and LVH- groups. The LVH+ group had higher SBP level, hs-CRP and sLOX1 levels than the LVH- group (P < 0.001).

Table 2 summarizes the genotype and allele frequencies of the LOX1 polymorphisms in two groups. Allele frequencies of the LOX1 polymorphisms among the EH patients were in Hardy-Weinberg equilibrium (all P>0.05). The genotype and allele frequencies of the intron 4 G>A and 3′UTR T>C SNPs were not significantly different between the LVH+ and LVH- groups (P>0.05). However, the genotype and allele frequencies of the 501G>C SNP were significantly different between those two groups. LVH+ patients had a markedly higher frequency of CC genotype than LVH- patients (60% vs.49 %, P=0.007). With the 501GG genotype as reference, multivariate logistic regression analysis showed that the 501CC genotype carriers had a markedly higher risk to develop LVH (adjusted OR=2.444, 95% CI:1.39-4.28, adjusted P=0.002) after adjustment for age, sex, alcohol intake, smoking status, BMI, TG, TC, HDL ,LDL, sCr , BP levels, hs-CRP and sLOX1 levels. The C allele carriers represented a significantly lower risk of developing LVH after adjustment of the above mentioned clinical variables (adjusted OR=1.571, adjusted P<0.001, Table 2) compared with the T allele carriers. In contrast, the multivariate logistic regression analysis did not reveal any association between of the SNPs, intron 4 G>A and 3′UTR T>C, and the occurrence of LVH (all P>0.05).

The associations between the LOX1 haplotypes and the LVH status were also explored in this study. The D´ value for the studied 3 SNPs was calculated using the SHEsis software. All 3 SNPs were in strong LD (all D´>0.8). The estimated haplotype frequencies of the LOX1 SNPs are shown in Table 3. The haplotype (intron4-501-3'UTR) A-C-C and G-C-T showed significantly higher association with the development of LVH (Table 3).

The association between the LOX1 gene polymorphism and the levels of sLOX1 and serum hs-CRP was studied. We found that only the 501G>C polymorphism was significantly correlated with the levels of sLOX1 and hs-CRP in this study. The 501CC genotype carriers had markedly increased levels of sLOX1 and hs-CRP compared to the 501GG and 501GC carriers (all P<0.001, Fig. 1).

We next studied the correlation between the levels of sLOX1 and hs-CRP, and the LVMI of EH patients. The LVH+ groups had a significantly higher mean serum level of LOX1 and hs-CRP compared to those without LVH (Table 1). The Pearson correlation analyses showed a positive correlation between the serum LOX1 levels and LVMI among enrolled subjects (r=0.907, P<0.001); the hs-CRP levels were also closely related to LVMI (r=0.876, P<0.001). In addition, there was also a strong correlation between the serum LOX1 levels and the hs-CRP levels among all enrolled subjects (r=0.862, P<0.001).

We then constructed the ROC curves based on the sLOX-1 and hs-CRP levels among the EH patients. The areas under curves (AUC) were 0.94 (95% CI: 0.89 -0.98, P<0.001) for sLOX-1 and 0.72 (95% CI: 0.68-0.79, P=0.021) for hs-CRP, respectively. A cutoff value of 1.0 ng/mL for sLOX-1 was able to divide the LVH+ patients from the LVH- patients, with 84% sensitivity and 86% specificity. In contrast, serum hs-CRP could not significantly differentiate the LVH+ and LVH- patients with a high specificity (74%), but lower sensitivity (45%) at a cutoff value of 3.6g/mL.

In the present study, we explored the correlations of the LOX1 gene polymorphisms and the serum LOX1 levels with the development of LVH among Chinese EH patients. We found that the 501>C polymorphism of the LOX1 gene and the serum LOX1 level were significantly associated with the risk to develop LVH. Our data suggest that both the 501>C of the LOX1 gene and the serum LOX1 level may be used as a potential marker for LVH incidence among the EH patients.

OLR1 is induced by proatherogenic stimuli and inflammatory cytokines [30]. Increasing evidence has indicated the critical roles of systematic vascular inflammation in the occurrence and progression of hypertension and atherosclerosis [27,31,32,33]. Polymorphisms of the OLR1 gene have been reported in association with EH, myocardial infarction (AMI) and coronary artery disease (CAD) [28,30,34]. Hou et al. has previously reported that the OLR-1 polymorphism, 501 G>C, was related to the severity of albuminuria among the EH patients [28]. The LOX1 501 G>C SNP is located in the open reading frame of the LOX1 gene, resulting in a mis-sense mutation of Lys to Asn at the amino acid position 167 (K167N) [29]. Functional studies have shown that the single amino acid replacement causes a significant reduction in ligand binding activity to Ox-LDL in the binding domain of LOX1 [35]. The LOX1 3'UTR C>T SNP is located at the 3' untranslated region of mRNA that has been shown to modulate the regulation of gene expression [27]. Its correlation to severity of AMI and CAD has also been proposed according to a number of research studies [27,33,34]. The intron 4 G>A SNP has been previously studied due to its occurrence in linkage disequilibrium (LD) with the 501 G>C and 3'UTR C>T SNPs in the OLR1 gene [30]. The association of polymorphisms of the OLR1 gene and risks to develop vascular diseases, however, is still inconclusive. The 501G>C SNP, for example, failed to show correlation in the development of the ischemic cerebrovascular diseases (CVD) [36]. Our study was not able to establish the statistical association between the SNPs, Intron 4 and 3'UTR, and LVH in the EH patients. This may be due to the size of sample that did not reach the power of calculation.

In line with a report from Chen et al., three SNPs we studied were in significant LD and were presented with similar allele frequencies (i.e., Intron 4>G 48.3%, 501 >G 48% and 3'UTR >T 48%). These frequencies are comparable to data generated from studies on non-Hispanic white populations, neverthelss, vastly different from the African American populations [27]. Evidence of LD has been widely reported between the polymorphisms of the LOX1 gene. Three common haplotypes account for 85% of the observed OLR1 haplotypes among the Centre d'Etude du Polymorphisme Humain (CEPH) individuals, who are representative of the European American population from the HapMap project (10 SNPs) [37]. Three LD blocks have been described [30]. Here, we, for the first time, report the similar allele frequencies of the intron 4 G>A, 501 G>C and 3'UTR C>T SNPs in the LOX1 gene between the Chinese population and certain Western population. In addition, the haplotypes (intron 4-501-3'UTR), A-C-C and G-C-T, showed significant correlation to the development of LVH in EH patients, which is consistent with the finding that the LVH+ patients had a markedly higher frequency of the CC genotype compared to the LVH- patients (P=0.007). This may provide evidence of using the 501 G>C SNP as a solo genetic marker to monitor the EH patients for their risk to develop LVH.

We also found that the LOX1 501CC genotype carriers had significantly increased sLOX1 and hs-CRP levels compared to the 501GG and 501GC carriers. Furthermore, not only the sLOX but also the hs-CRP levels were significantly higher in the LVH+ group compared to the LVH- group. The sLOX-1 level, however, may be a better parameter than hs-CRP to differentiate the LVH+ patients from the LVH- patients based on the ROC curves. These experimental results correlate to close functional partnership of LOX1 and CRP that has been well studied in their pro-atherogenic effects on vascular cells [16,38,39,40,41]. Among all, a recent study suggested that L5, a subfraction of plasma LDL, CRP and the LOX-1 may constitute a positive feedback loop that contributes to endothelial dysfunction [39]. LOX-1 along with Fcγ receptors functions as a receptor for CRP [42]. CRP elicits oxidative stress and compromises vasomotor function on porcine coronary arterioles via LOX-1 activation [38]. In addition, CRP promotes the release of soluble LOX-1 from macrophages by activating tumor necrosis factor alpha (TNF-α) converting enzyme [39] and increases vascular permeability through direct binding to LOX-1[42]. On the other hand, LOX-1 mediated upregulation of a thrombogenic protein, plasminogen activator inhitor-1 (PAI-1) in arterioles by CRP [38]. LOX-1 may be also involved in CRP-induced complement activation, and thus may serve to locate the site of CRP-induced complement activation and inflammation [42]. Those emerging pieces of evidence could facilitate our understanding to the mechanisms involved in the regulation of the LOX1 501 SNP on the sLOX1 and hs-CRP levels among EH patients.

Several limitations should be addressed in this study. Firstly, this is a single center study on the Chinese population. A validation cohort is necessary to confirm the association between the serum LOX1 levels and the LOX1 SNPs among EH patients with LVH. Secondly, the findings of this study only indicate the association between the LOX1 polymorphisms and the serum LOX1 levels among EH patients with already developed LVH. Ideally, the longitudinal echocardiography data is required to determine if a genotype as a biomarker is predictive of the progression and development of LVH in EH patients.

Taken together, data from our study indicates that the LOX1 501 >C SNP as well as the LOX1 haplotypes, A-C-C and G-C-T, which carry this particular SNP, are significantly associated with the incidence of LVH among the EH patients. The sLOX-1 level may also serve as a biomarker to monitor EH patients in the development of LVH. Both the prospective and larger scale studies are warranted to verify our findings in order to apply into clinical practice.