Our Study Revealed a Strong Association of Apolipoprotein E Locus with Essential Hypertension

Our Study Revealed a Strong Association of Apolipoprotein E Locus with Essential Hypertension

Abstract

Hypertension, a state of oxidative stress is associated with Nitric oxide – Ang II imbalance that may be an important component in pathophysiology of hypertension.Recent experimental data suggests marked similarities between the effects of hypertension and hypercholesterolemia, on the arterial intima. Endothelial dysfunction is in the cross road between hypertension anddyslipidemia. Apolipoprotein E , a polymorphic protein encoded by 3 alleles -  2, 3 & 4, has antioxidant, inhibition of smooth muscle proliferation,inhibition of lipid peroxidation and nitric oxide generating properties in addition to anti-atherogenic effects, which are isoform dependent.

In this case – control study it was proposed to study the association of apolipoprotein E polymorphism and essential hypertension.The Apolipoprotein E genotyping was done on 155 hypertension patients and 152 controls by polymerase chain reaction and subsequent digestion with Hha1.

Incidence of 4 allele was significantly higher in patients (59.4 %) than in controls (25%; p=0.000).Multiple logistic regression analysis indicated that4 allele is an independent predictor of HT. There was no significant difference between 4 allele carriers and non-carriers with regard to total cholesterol and low density lipoprotein cholesterol levels in patients ,whereas in controls total cholesterol and low density lipoprotein cholesterol levels were higher in 4+ allele than in 4 – allele carriers. The odds ratios for 4 and 2 alleles versus 3allele were 4.79 and 2.08 respectively.

Our study revealed a strong association of apolipoprotein E locus with essential hypertension.

Key words: nitric oxide, angiotensin II, oxidative stress, cardiovascular disease

INTRODUCTION

Cardiovascular diseases (CVDs) will be the largest cause of death and disability by 2020 in India,according to WHO report. The Global Burden of Disease (GBD) study estimates that 52% of CVD deaths occur below the age of 70 years in India, resulting in a profound adverse impact on its economy. The contributing factors are increasing prevalence of cardiovascular risk factors especially hypertension, dyslipidemia, diabetes, overweight or obesity, physical inactivity and tobacco use. (1)

Hypertension prevalence has been increasing and an estimated 972 million people in the world are suffering from this problem(2). Relationship between blood pressure and risk of cardiovascular events is continuous, consistent and independent of other risk factors(3)

Hypertension , a disease largely of the vasculature, is a multifactorial disorder with genetic contributions ranging from 30% to 60%(4).

The potential mechanisms of hypertension that constitute aberrations of normal regulatory processes also involves increased formation of ROS, particularly hydrogen peroxide(5) and an excessive activity of renin – angiotensin – aldosterone system which contributes to endothelial dysfunction, vascular inflammation and thrombosis(6,7).

Angiotensin1 receptor(AT1) activation leads to production of ROS in the vessel wall, in part because the AT1 receptor is linked to activation of an NADH/NADPH (NAD[P]H) oxidase in vascular cells(8,9). Impaired endothelium-mediated vasodilation in hypertension has been linked to decreased NO bioavailability(9).The development and/or maintenance of both hypertension and the abnormal vascular remodeling is probably due, in part to a loss of NO and, more important, to an imbalance among Ang II, NO, and O2 production(10,11,12).

Recent experimental data suggests marked similarities between the effects of

hypertension and hypercholesterolemia, on the arterial intima(13). Endothelial dysfunction is in the cross road between hypertension and dyslipidemia(6).The key feature is a reduced availability of nitric oxide (NO). Hypercholesterolemia diminishes the expression of endothelial NO synthase(6) and stimulates the activity of plasma and tissue renin and the subsequent production of angiotensin II,which causes an increase in the activity of the angiotensin II type 1 receptor, which in turn potentiates the effects of hypercholesterolaemia on renin activity(14).

In the setting of endothelial dysfunction, there is an increase in vascular permeability to LDL, which becomes oxidized in the arterial wall where the macrophages uptake them evolving into foam cells. These processes are promoted by AT1 receptor activation and the expression of the oxidized LDL receptor LOX is enhanced through AT1 activation (6).

The plasma concentration of lipoproteins and their metabolic fates are modulated by apolipoproteins on the surface of these lipid rich particles. It has been hypothesized that genetic variation in apolipoproteins is a major determinant of the interindividual variation in susceptibility to CAD(15).

Apolipoprotein E ,a member of the apolipoprotein gene family with its gene located in chromosome 19q 13.2 , produces a 299 aminoacid polypeptide(16,17). It is coded by 3 common alleles-2,3, 4 producing 3 isoforms of protein E2, E3 and E4 (18, 19).

Apo E gene polymorphism has a strong effect on the level of its gene product, functions and catabolism .2 are associated with higher concentation of apo E and4 with lower concentration(18,20-22).

Apo E is a key protein in the modulation of the metabolism of highly atherogenic apo B containing lipoproteins(23). Apo E3 is the predominant apo E isoform in the normolipidemic population and 3 has been proposed to be the normal allele.

Apolipoprotein E is a protein with multiple hats (24,25). Apo E has anti atherosclerotic effect(24), Antioxidant(24,26), Antiproliferative (smooth muscle cells, lymphocytes)(24,27), Anti-inflammatory(24), Antiplatelet(28), and Nitric oxide (NO) generating properties(29,30) which are allele specific.

The three alleles of apolipoprotein E predict varying degrees of risk / protection for certain diseases because of their isoform specific differences in their functions.Protection conferred by apoE3 is its ability to attenuate TNF-α induced inflammatory responses, to modulate endothelial NO and in the vascular wall to balance the intracellular redox state in injured endothelial cells via NO-dependent and independent pathways.4 allele is associated with higher total cholesterol and low density lipoprotein cholesterol and is considered “proatherogenic”. Apo E4 has decreased antioxidant activity, NO production and least effective in promoting cholesterol efflux from macrophages and leads to increased atherosclerosis. 4 isoform is proinflammatory through direct oxidant properties or through deficits in NO signaling(25).

As Apolipoprotein E has properties capable of counteracting the ROS mediated biochemical insults on arterial wall, it is hypothesized that apolipoprotein E polymorphism could be a genetic marker in the development of hypertension.

The aim and objectives of the current study are to find an association between apolipoprotein E polymorphism and essential hypertension and to correlate it with plasma lipid levels,to study the distribution of apo E alleles in the population being studied and to study the effects of ε -4 allele with hypertension and plasma lipid levels.

MATERIALS AND METHODS:

This case – control study was carried out in the Hypertension Clinic, Department of Internal Medicine, Madras Medical college and Government General hospital, Chennai-03 during the period April 2008 – October 2008.Study group consisted of 155 cases with essential hypertension and 152 hospital based patients who were free of hypertension as controls. Patients with secondary hypertension and those with renal disorders were excluded.The evaluation of confounding risk factors to the development of essential hypertension was based on personal history, physical examination and laboratory. findings. Control subjects were also similarly evaluated .The confounding risk factors included smoking, alcohol consumption, dyslipidemia and family history of hypertension.

Hypertension was considered to be present if an individual had a history of hypertension or was on antihypertensive agents or if the systolic blood pressure exceeded 140mmHg or the diastolic pressure exceeded 90mmHg( 3).Informed consent was obtained from all the study participants and the study was approved by institutional ethics committee.

4ml of peripheral venous blood was withdrawn from all the study subjects after 12 hours fasting, under sterile conditions in KF: Na2EDTA tubes. Buffy coat was separated by centrifugation of KF: Na2EDTA tubes at 2000 revolutions for 20 minutes. Buffy coat was transferred to 2ml eppendorf and was used for DNA extraction.

Plasma separated was used for lipid profile estimation.Total cholesterol (TC), high density lipoprotein cholesterol (HDL-c) andtriglyceride concentration (TGL) were determined enzymatically using Transasia Laboratories kits and XL-300 auto analyzer at Centralized Biochemistry Laboratory at Government General Hospital, Chennai-3.Low density lipoprotein cholesterol (LDL-c) was calculated using Friedwald’s formula.

DNA was extractedby Modified High Salt Method (31).Concentration of extracted DNA was estimated using UV spectroscopy at 260nm. Purity of extracted DNA was assessed by 260/280 ratio and it was found to be > 1.7.Apolipoprotein E gene was amplified using the extracted DNA as template and the following forward and reverse primers { Forward (5’ACAGAATTCGCCCCGGCCTGGTACAC-3’) and Reverse

(5’TAAGCTTGGCACGGCTGTCCAAGGA-3’) primers} in an Applied Biosystems thermal cycler with the following cycling conditions.• Initial denaturation – 95*C -5min and • 30 cycles of - Denaturation – 95*C – 1 min, Annealing - 60*C – 1min, Extension - 70*C – 2min, • Final extension at 72*C - 10 min(32). Amplicons of 244 bp was identified by 2% agarose gel and was subjected to restriction digestion with HhaI enzyme. Restriction digested product was subjected to PAGE for genotyping.PAGE was performed on 22% gel with digestedfragments and genotype was identified based on the presence of base pairs as-E2/2 91 & 83 bp, E3/3 91, 48 & 35 bp, E4/4 72, 48 & 35 bp, E3/4 91,72,48 & 35 bp, E2/4 91,83,72,48 & 35 bp, E2/3 91,83,48 & 35 bp.(figure : 1)

Standard statistical procedures from the SPSS package were used for the analysisof the data. For major risk factors and potential confounders, differences between hypertensive cases and control groups were tested by using appropriate tests of significance (2, and t test).Odds ratios were calculated for apo 4 allele (4/4and 3/4) and 2 allele (2/2, 2/3 and 2/4) taking 3 allele (3/3) as reference. For each odds ratio two tailed p values and 95% confidence intervals (CI) was calculated. Significance levels were set at 0.05 in all cases.

RESULTS:

In this study, 155 patients with essential hypertension were comparedwith 152 normotensive controls.The mean age of patientswas 55±9.75 years, while in controls it was44.3±12.39 years (p=0.00)(Table 1). SBP (140.7±19.89mmHg, p=0.00) and DBP (87.61±12.64 mmHg, p=0.00) were found to be higher in patients than in controls (120.37±11.26 mmHg and 81.18±8.69 mmHg, p=0.00) respectively. Patients showed a strong association with positive family history (37.4% in patients vs. 9.9 % in controls,2=32.14, p=0.00), as compared to controls.

The distribution of apo E genotypes in hypertensives differed significantly from controls ((2=38.73, p=0.0).There was no significant difference in genotype distribution in males vs. females and familial vs. non-familial cases.

E3/4 genotype was 2.4 times high in patients than controls (54.2% vs. 22.37%,p=0.0) and E3/4 genotype was the predominant genotype in patients. E 2/3 genotype was high in controls (7.24%vs 6.5%) than in patients. E3/3 was the predominant genotype in controls(67.8% vs 34.2%). E2/4 & E4/4 genotypes were also associated with patients than in controls (E2/4- 3.2% vs. 1.97%, E4/4 – 1.9% vs. 0.66%)(table :2)

4-allele frequency was more prevalent in patients than in controls (59.3% vs 25.1%,2 = 35.12, p<0.05). 3 allele frequency was more prevalent in controls than in patients (68% vs. 34%,2 =34.64, p<0.05), while2 allele did not show any difference(patients 6.5% vs 7.2% in controls) . The allelic distribution was significantly deviated from Hardy – Weinberg equilibrium in both patients and controls with regard to 3 and 4 alleles.

It was found that the presence of 4 allele conferred a 4.2 fold higherrisk for the development of hypertension as compared to the other 2 alleles (0.R=4.2). Also when 3 allele was compared with 4 allele (O.R=0.4) it was found to confer protection against the development of hypertension.

Mean distribution of lipid levels in patients and controls showed no difference with regard to TC, LDL-c, HDL-c and TGL.When patients and controls were divided based on 4 allele status, patients did not show any difference in their lipid profile between 4 allele carriers and non-carriers. Where as in controls TC and LDL-c levels were higher in 4+ allele than in 4- allele (p=0.00). HDL-c levels were higher in 4 – allele (p=0.00),indicating the effect of apo E locus on lipid metabolism.( table :3)

Multiple logistic regression analysis indicated that 4 allele is a significant and independent predictor of hypertension. Odds ratio for 4 and 2 alleles vs. 3 allele as reference were 4.79 and 2.08 respectively (table: 4 )

DISCUSSION:

Gene frequencies at apo E locus are highly heterogeneous in populations(21). InIndia there has been a high prevalence of 3 allele towards north, 2 and 4 allele frequencies decline from south towards north(33). Population based study in South India reported an allele frequency of 85-92% - 3 allele , 3-9% 4 allele and 3-5% 2 allele(34). In the present study there has been a considerable difference with regard to allele frequencies both in cases and controls as compared with previous studies. 4 allele was more predominant in cases and 3allele in controls. But the percent distribution of 4 allele was higher in controls (23% vs. 3-9%) as compared with previous population studies. 4 allele by nature of its proatherogenic property has been attributed as an independent risk factor for CAD. But the association of 4 allele with hypertension has shown inconsistent results in previous studies(24). With the new perspective of

ROS mediated vascular injury in the pathophysiology of hypertension,the hypothesis that apo E by virtue of its ability to balance the intracellular redox state in injured endothelial cells via NO dependent and independent pathways has been implicated to be associated with essential hypertension.

The present study confirms a strong association of apo E locus (4 allele) to

essential hypertension. The reasons in favor of the hypothesis are:

1. Allele specific antioxidant activity of apo E (E2>E3>E4) .E4 exhibits least

antioxidant activity .

2. NO production by apo E is isoform dependent. Apo E2 producing half that of E3 while E4 fails to increase NO levels above the control (29,30).

3. 4 allele is proatherogenic and proinflammatory (35,36) being associated with higher TC and LDL-c levels and lower HDL-c levels, which predisposes to impaired vascular remodeling and thus endothelial dysfunction. It is proinflammatory through direct oxidant property or through deficits in NO signaling.

4. Apo E4 isoform is associated with lower apo E levels .

5. Carotid intima- media thickness correlate with blood pressure levels and apo E is associated with mean intima – media ratio reduction ( 37)

The above factors predict a strong association of 4 allele with hypertension. Tofurther assess the antioxidant and NO producing property of 4 allele,plasma apo E levels could be measured . iPF2a-VI , an isoprostane – an accepted index of in vivo oxidant stress, along with NO levels in plasma can help in predicting association of 4 allele with hypertension.

With regard to lipid profile in patients and controls, the lack of association was

due to patients being treated with statins, which abolished the difference(38). Also

control group had higher percentage of smokers and alcoholics as compared to

patients. Statins have proved to reduce mortality to a greater extent in those with 4 allele. Previous studies have revealed that LDL-c response to lovastatin was

significantly lower in 4 group compared with 2 and 3 groups(37,39).

Further it has been shown that plant sterol and cholesterol absorption is increased in E3/4 and E4/4 genotypes when compared to those with E3/3 genotype(40). This increased absorption is not associated with a decrease in the synthesis of cholesterol. These differences could influence the efficacy of drugs that inhibit the absorption or synthesis of cholesterol. Within the control group it was observed that the TC and LDL-c levels were significantly higher in 4 (+)allele carriers as compared to 4(-) allele non -carriers. Also there was higher HDL-c levels in 4(-) allele non –carriers which proved the association of apo E locus with lipid metabolism.

CONCLUSION :

The association of 4 allele with hypertension to be confirmed, needs concurrentquantitative assessment of isoprostane- iPF2-VI levels (in vivo oxidant stress marker),plasma NO andapo E levels .Apo E mediates its adverse or protective actions via severalmechanisms, which assume variable degrees of relevance depending on factorslike ethnicity, environment and treatment.

Apo E genotyping can be used for screening as well as prediction of response to a specific treatment. As 4 allele has been predicted as a significant risk factor for CAD, intensive life style modification and pharmacological therapy to these individuals may bring down hypertension and dyslipidemia associated risk of CAD. The role of apolipoprotein E in therapeutics, if proved can be a boon to individuals with apoE polymorphism( especially for 4 allele individuals ).

Acknowledgements

I thank Dr .Kumanan Professor and HOD, Department of Animal Biotechnology, Veterinary college Chennai, for permitting me to carry out the molecular work in their laboratory.

I thank my Mother Dr.Savithri Govindharajulu for funding this project which was done as part of my MD dissertation work

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