Introduction
Hypertension has been recognized as a significant
risk factor for the development of cardiovascular diseases,
including coronary heart disease (CHD), myocardial infarction (MI)
and stroke, all of which are principal causes of cardiovascular
morbidity and mortality in humans. Essential hypertension (EH) is a
complex, polygenic disease in which numerous genes control the
blood pressure level. Several candidate genes, all selected from
the renin-angiotensin system-related genes, have been examined. One
of the candidate genes is the gene encoding angiotensinogen (AGT),
the precursor of the vaso-active peptide, angiotensin II. The
angiotensin I-converting enzyme (ACE) gene has also been associated
with EH in the majority of the population. In recent years, the
correlation between the molecular variant of the AGT gene, M235T,
and EH has started to receive attention (1–6). AGT
in the circulation is mainly synthesized in the liver. The plasma
AGT level mainly reflects this synthesis. It has been discovered
that the expression of the AGT gene in the liver is regulated by
certain cytokines. It has been reported that these cytokines
[interleukin (IL)-1, IL-6 and tumor necrosis factor-α (TNF-α)] are
likely to be the main activation factors in AGT gene expression.
They activate the expression of the AGT gene, elevate the
concentrations of AGT and ultimately accelerate the development of
hypertension. In addition, IL-1 and TNF-α also induce proliferation
of the smooth muscle blood vessel cells and arteriosclerosis.
Studies on the association between the M235T polymorphism of the
AGT gene and cytokines in hypertensive Chinese patients have been
rarely reported. The aim of this study was to analyze the M235T
polymorphism of the AGT gene and cytokine levels in Chinese
patients with EH and compare them to healthy controls. Such a
complex study is the first to be carried out in China.
Materials and methods
Hypertensive patients
The study group consisted of 300 patients with EH.
All patients were hospitalized in the Department of Cardiology,
First Affiliated Hospital of Anhui Medical University, China and
included 120 males (40%) with a mean age of 55.11±8.41 years and
180 females (60%) with a mean age of 58±10.19 years. Risk factors
for EH were obtained by standard questionnaires, physical
examinations and blood tests. The patients were asked about their
medical history, family history of hypertension, smoking habit and
alcohol consumption. Blood pressure was measured by standard
methods. All patients had an onset of hypertension prior to the age
of 50 years. We considered patients to have EH if they had a
systolic blood pressure (SBP) above 140 mmHg and/ or a diastolic
blood pressure (DBP) above 90 mmHg at the time of examination, and
no clinical evidence of secondary hypertension.
Healthy controls
The healthy control group consisted of 150
age-matched individuals. The group of normotensive individuals
included 55 males (36.67%) with a mean age of 51.55±20.89 years and
95 females (63.33%) with a mean age of 53.16±12.26 years. None of
the individuals from the control group had symptoms of EH. They
each had an SBP lower than 140 mmHg and a DBP lower than 90 mmHg.
None of the control group subjects had a positive familial history
of EH or MI. None of these individuals had secondary hypertension
or diabetes. All the individuals in the control group had never
been treated with anti-hypertensive medication. All EH patients and
healthy controls provided informed consent, and responded to a
questionnaire that provided information concerning risk factors of
hypertension, including alcohol consumption, cigarette smoking and
familial history of hypertension. The study and DNA analysis were
approved by the ethics board of our university. The main
characteristics of the two groups are presented in Table I.
 | Table I.Main characteristics of the
hypertensive group and the healthy controls. |
Table I.
Main characteristics of the
hypertensive group and the healthy controls.
| Characteristics of
cases (n) | Hypertensive group
(n=300) n (%) | Healthy controls
(n=150) n (%) |
|---|
| Female | 180 (60.0) | 95 (63.3) |
| Mean age,
years | 58.0±10.2 | 53.2±12.3 |
| Alcohol
consumption | 16 (8.9) | 15 (15.8) |
| Cigarette smoking
(at least 10 cigarettes daily) | 15 (8.3) | 7 (4.7) |
| Familial history of
hypertension | 68 (37.8) | 0 (0) |
| Male | 120 (40.0) | 55 (36.7) |
| Mean age;
years | 55.1±8.4 | 51.6±20.9 |
| Alcohol
consumption | 25 (20.8) | 28 (50.9) |
| Cigarette smoking
(at least 10 cigarettes daily) | 50 (41.7) | 15 (27.3) |
| Familial history of
hypertension | 94 (78.3) | 0 (0) |
Blood pressure measurement
Blood pressure was measured using a mercury-gravity
manometer. Measurements were recorded using the left arm with
subjects in the seated position following 10 min of resting. This
procedure was repeated three times, and the systolic and diastolic
blood pressures were defined as the means of the three independent
measurements.
Materials
The primers for polymerase chain reaction (PCR) were
purchased from the Institute of Biochemistry and Cell Biology,
Shanghai, China. Taq enzymes were provided by HuaMei Co., Shanghai.
The cytokine determination reagent boxes of IL-1, IL-6 and TNF-α
were provided by Genezyme Co., Cambridge, MA, USA.
DNA isolation and PCR-restriction
fragment length polymorphism (RFLP)
In order to identify the M235T polymorphism of the
AGT gene, 3 ml of venous blood was drawn into tubes containing 0.3
ml of 2% EDTA, and was centrifuged for 10 min at 5,000 rpm. The
blood plasma was separated and preserved at −20°C, and the surplus
blood cells were mixed with 20 ml of distilled water. The red blood
cells were hemolyzed after 15 min and were centrifuged for 10 min
at 5,000 rpm. The precipitated white blood cells, without the
supernatants, were preserved at −20°C. The white blood cell DNA was
then digested with Proteinase K, extracted with phenol and
chloroform, and then preserved at −20°C. The analysis took 3
months.
PCR-RFLP was used to examine the M235T polymorphism
of the AGT gene. The primers used to amplify the DNA fragment (98
bp) were as follows: primer 1 (627–646 nt), 5′-AGA ACT GGA TGT TGC
TGC TG-3′; and primer 2 (724–705 nt), 5′-TGC TGT CCA CAC TGG CTC
GC-3′. PCR was carried out in a 50 μl volume with 35 cycles of
amplification (92°C for 44 sec, 55°C for 40 sec and 72°C for 90
sec) with a final extension at 72°C for 10 min. The M235T
polymorphism of the AGT gene was detected by the digestion of the
PCR products with a BstUI restriction enzyme, and the
digestion was performed in a 10 μl reaction mixture. The digested
products were separated by electrophoresis on a 2% agarose gel or
7% PAGE with silver staining. The positive M235T polymorphism of
the AGT gene was confirmed by DNA sequencing (data not shown).
Enzyme-linked immunosorbent assay
(ELISA)
This was used to detect the concentrations of the
cytokines (IL-1, IL-6 and TNF-α). The determination reagent boxes
of IL-1, IL-6 and TNF-α were as follows: i) gene recombination
IL-1, purity >98%, 2x107 u/mg; ii) gene recombination
IL-6, purity >96%, 2x107 u/mg; iii) gene
recombination TNF-α, purity >96%, 1.5x107 u/mg. The
determination using microELISA was performed according to the
manufacturer’s instructions.
Statistical analysis
All the data were expressed as the means ± SD.
Statistical analyses of the differences between the two groups were
performed using the homogeneity test for variance and the unpaired
data t-test. The intergroup difference was judged to be significant
at P<0.05, and was considered to be greatly significant at
P<0.01.
Results
Hardy-Weinberg equilibrium
The distribution of the AGT gene 235T/M genotype in
the hypertensive and healthy control group subjects, recruited from
city of Hefei in China, was analyzed using the Hardy-Weinberg
equilibrium (χ2=0.032, P>0.05).
AGT gene 235TT, TM and MM genotypes
Three genotypes (TT, TM and MM) were detected in the
235 codon of the AGT gene. In the hypertensive group, the
percentages for the AGT gene TT genotype, MT genotype and MM
genotype were 55, 36 and 9%, respectively. The ratio of the T/M
allele frequency was 0.73/0.27. In the control group, the
percentages for the AGT gene TT genotype, MT genotype and MM
genotype were 46, 44 and 10%, respectively. The AGT ratio of T/M
allele frequency was 0.68/0.32. In addition, the AGT gene 235TT
genotype and T allele frequencies in the hypertensive group were
significantly higher than those in the control group (P<0.05)
(Table II).
| Table II.M235T AGT genotypes in the
hypertensive group and the healthy controls. |
Table II.
M235T AGT genotypes in the
hypertensive group and the healthy controls.
| Hypertensive
group | Healthy controls |
|---|
| No. of cases | 300 | 150 |
| TT genotype, n
(%) | 165 (55) | 69 (46) |
| MT genotype, n
(%) | 108 (36) | 66 (44) |
| MM genotype, n
(%) | 27 (9) | 15 (10) |
The correlation between T and M allele
frequency and the cytokine concentrations
The correlation between the T and M allele frequency
and the cytokine concentrations (IL-1, IL-6 and TNF-α) in the two
groups was compared. There was a significant difference in the
average concentrations of cytokines (IL-1 and TNF-α) in the
patients with the AGT gene 235T allele between the hypertensive
group and the healthy control group (P<0.01). However, there was
no significant difference in the average cytokine concentrations of
(IL-1, IL-6 and TNF-α) in the patients with the AGT gene 235M
allele between the two groups (P>0.05) (Table III).
| Table III.T and M allele frequencies for the
hypertensive group and the healthy controls and the cytokine
concentrations (IL-1, IL-6 and TNF-α) (means ± SD). |
Table III.
T and M allele frequencies for the
hypertensive group and the healthy controls and the cytokine
concentrations (IL-1, IL-6 and TNF-α) (means ± SD).
| Hypertensive group
(300)
| Healthy controls
(150)
|
|---|
| Group | T (73%) | M (27%) | T (6%) | M (32%) |
|---|
| IL-1 (ng/ml) | 13.4±3.8a | 9.4±2.8b | 10.8±3.3 | 8.7±3.3 |
| IL-6 (ng/ml) | 3.9±1.3b | 5.8±2.7b | 5.6±2.4 | 4.4±3.2 |
| TNF-α (ng/ml) | 11.4±2.6a | 10.4±3.5b | 7.7±2.2 | 9.3±2.3 |
The blood concentrations of IL-1, IL-6
and TNF-α
There were significant differences in the blood
concentrations of IL-1 and TNF-α between the patients in the
hypertensive group and those in the healthy control group
(P<0.01), but there was no difference in IL-6 (P>0.05)
(Table IV).
| Table IV.Cytokine (IL-1, IL-6 and TNF-α)
concentrations in the hypertensive group and the healthy controls
(means ± SD). |
Table IV.
Cytokine (IL-1, IL-6 and TNF-α)
concentrations in the hypertensive group and the healthy controls
(means ± SD).
| Hypertensive
group | Healthy
controls |
|---|
| No. of cases | 300 | 150 |
| IL-1 (ng/ml) | 12.9±3.6a | 9.5±3.0 |
| IL-6 (ng/ml) | 4.5±2.5b | 5.0±2.9 |
| TNF-α (ng/ml) | 11.2±3.0a | 7.91.9 |
Discussion
This study explored the association between the
M235T polymorphism of the AGT gene and cytokine concentrations
using age-matched individuals with different backgrounds. In this
way, our results were more concise compared to those from studies
using stochastic control groups.
In the last few years, the M235T polymorphism of the
AGT gene has been investigated for an association with EH, based on
conventional measurements of blood pressure. However, the results
have been inconsistent. There have been a number of studies that
have confirmed a positive association between M235T variants and
hypertension (6–8), but certain studies have confirmed a
lack of significant effect of the AGT M235T polymorphism (9,10).
This means that different populations may have a different AGT
M235T polymorphism.
Previously, an extensive study on the potential role
of the AGT gene in EH was performed by Jeunemaitre et al in
two large series of 379 sibling pairs. They found genetic linkage
between EH and AGT in the affected siblings and an elevated M235T
allele frequency, compared to healthy controls. In the second year
of this study, results from the same group confirmed an association
between the M235T variants and EH (11).
Another study conducted in a Japanese population of
352 individuals, with a mean age of 52.5 years, reported no
association between the M235T variant of the AGT gene and EH
(12). Rotimi et al studied
the association between EH and the M235T variant in the
African-American population. They revealed that the frequency of
the 235T allele was 83% in hypertensive patients and 82% in control
subjects. These results offered no evidence of a linkage between
the 235T allele and EH in the African-American population (13).
The variety in these results could be due to
differences in ethnicity. The results of our study suggest an
association between the M235T polymorphism in the gene encoding AGT
in Chinese patients with EH. Three genotypes (TT, TM and MM) were
separated from the 235 codon in the AGT gene using PCR-RFCP. Among
the 300 Chinese patients with EH, 36% were heterozygous (genotype
TM) and 55 and 9% were homozygous (genotype TT and MM); while among
the healthy Chinese controls, 44% were heterozygous (genotype TM)
and 46 and 10% were homozygous (genotype TT and MM) (Table II). In the hypertensive group, the
ratio of the T/M allele frequency was 0.73/0.27. In the control
group, the ratio of the T/M allele frequency was 0.68/0.32. The AGT
gene 235TT genotype and T allele frequencies in the hypertensive
group were significantly higher than those in the control group.
This was a preliminary study and more cases are required for
further study. Further investigation is required to clarify the
potential involvement of the M235T polymorphism.
We also discovered that the blood concentrations of
the cytokines (IL-1 and TNF-α) in the hypertensive group were
markedly higher than those in the control group (P<0.01), while
there was no significant difference in IL-6 concentration between
the two groups (P>0.05). In addition, there were significant
differences in the average concentrations of IL-1 and TNF-α in the
AGT gene 235T allele (P<0.01), but not in the AGT gene 235M
allele (P>0.05). Our study suggests that hypertension occurs and
becomes aggravated easily in individuals who are carriers of the
AGT gene T allele when the concentrations of cytokines (IL-1 and
TNF-α) in the carriers are elevated. High levels of IL-1 and TNF-α
may promote inflammation, higher expression of AGT and
Ca2+ movement into the vascular smooth muscle cells
(VSMC) in EH patients. EH is a type of inflammation-related
disease.
In conclusion, we consider that the AGT gene
moleculer variant, M235T, may be a significant risk factor and
hereditary marker for EH. The higher frequency of the AGT gene TT
genotype and the AGT gene 235T allele may promote the development
of hypertension. In EH patients with the AGT gene 235T allele,
elevated concentrations of IL-1 and TNF-α may enhance the
transcription and expression of the AGT gene and may cause the
blood vessels to constrict.
References
|
1.
|
LD JiLN ZhangP ShenAssociation of
angiotensinogen gene M235T and angiotensin-converting enzyme gene
I/D polymorphisms with essential hypertension in Han Chinese
population: a meta-analysisJ
Hypertension28419428201010.1097/HJH.0b013e32833456b920087216
|
|
2.
|
D MarkovicX TangM GurujuAssociation of
angiotensinogen gene polymorphisms with essential hypertension in
African-Americans and CaucasiansHum
Hered608996200510.1159/00008865716210856
|
|
3.
|
J YuanW TangY ChunAngiotensinogen T174M
and M235T variants and hypertension in the Hani and Yi minority
groups of ChinaBiochem
Genet47344350200910.1007/s10528-009-9237-319365726
|
|
4.
|
AD TiagoNJ SamaniGP CandyR
BrooksbankAngiotensinogen gene promoter region variant modifies
body size-ambulatory blood pressure relations in
hypertensionCirculation10614831487200210.1161/01.CIR.0000029093.93362.FC12234952
|
|
5.
|
SJ WuFT ChiangWJ ChenThree
single-nucleotide polymorphisms of the angiotensinogen gene and
susceptibility to hypertension: single locus genotype vs. haplotype
analysisPhysiol
Genomics177986200410.1152/physiolgenomics.00133.200314970360
|
|
6.
|
YJ FangHB DengGN ThomasLinkage of
angiotensinogen gene polymorphisms with hypertension in a sibling
study of Hong Kong ChineseJ Hypertens2812031209201020216084
|
|
7.
|
E LizaneczET PásztorA MohácsiMistyping of
angiotensiogen M235T allelesHypertens
Res29197201200610.1291/hypres.29.19716755155
|
|
8.
|
M CaulfieldP LavenderM FarrallLinkage of
the angiotensinogen gene to essential hypertensionN Engl J
Med33016291633199410.1056/NEJM1994060933023018177268
|
|
9.
|
N GlavnikD PetrovicM235T polymorphism of
the angiotensinogen gene and insertion/deletion polymorphism of the
angiotensin-1 converting enzyme gene in essential arterial
hypertension in CaucasiansFolia Biol536970200717448297
|
|
10.
|
M MettimanoA LanniA
MignecoAngiotensin-related genes involved in essential
hypertension: allelic distribution in an Italian population
sampleItal Heart J25895932001
|
|
11.
|
X JeunemaitreA CharruG ChatellierM235T
variant of the human angiotensinogen gene in unselected
hypertensive patientsJ Hypertens
Suppl11S80S81199310.1097/00004872-199312050-000198158445
|
|
12.
|
S IchiharaM YokotaT FujimuraLack of
association between variants of the angiotensinogen gene and the
risk of coronary artery disease in middle-aged Japanese menAm Heart
J134260265199710.1016/S0002-8703(97)70133-99313606
|
|
13.
|
C RotimiL MorrisonR CooperAngiotensinogen
gene in human hypertensionLack of an association of the 235T allele
among African-Americans Hypertension2459159419947960018
|
|
14.
|
AL WangDH KongDX ChenMutation of V896M in
cardiac myosin binding protein-c gene in two Chinese families with
hypertrophic cardiomyopathyMol Med Report3759763201021472310
|
