http://informahealthcare.com/bmk ISSN: 1354-750X (print), 1366-5804 (electronic) Biomarkers, 2014; 19(4): 314–318 ! 2014 Informa UK Ltd. DOI: 10.3109/1354750X.2014.910550
RESEARCH ARTICLE
Expression of angiotensin-converting enzyme gene in whole blood in patients with essential hypertension Sudhir Chandra1, Rajiv Narang2, Daman Saluja1, Jagriti Bhatia3, and Kamna Srivastava1#
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1
Dr. B R Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India, 2Department of Cardiology, and 3Department of Pharmacology, All India Institute of Medical Sciences, New Delhi, India Abstract
Keywords
Objective: The present study aims to investigate the correlation of the angiotensin-converting enzyme (ACE) gene expression and protein expression in patients with essential hypertension in whole blood. Methods: ACE gene expression was analyzed by Real Time PCR and western blot in 52 patients with essential hypertension and 42 healthy controls. Results: We observed a significant increase in Delta threshold cycle (DCT) values in the circulating ACE gene and ACE protein expression in patients as compared to controls. Conclusions: The up-regulation in relative expression of circulating Angiotensin converting enzyme mRNA and protein in patients with respect to controls might be correlated with high blood pressure in patients with essential hypertension.
Angiotensin-converting enzyme, blood pressure, essential hypertension, gene expression, renin–angiotensin–aldosterone system
Introduction Hypertension is major public health problem all over the world, including India. Essential hypertension is the form of hypertension that has no identifiable cause but it affects 95% of hypertensive patients (Carretero & Oparil, 2000; Hall & Guyton, 2006; Oparil et al., 2003) probably due to the consequence of an interaction between environmental and genetic factors. Angiotensin-converting enzyme (ACE), an important component of renin–angiotensin–aldosterone system (RAAS) converts angiotensin I into a physiologically active peptide angiotensin II, a potent vasopressor and aldosteronestimulating peptide that controls blood pressure and fluidelectrolyte balance (Carey et al., 2000). Several genes of RAAS such as Angiotensin II type 1 receptor (AT1R) (Wang et al., 2010), ACE insertion/deletion (ACE I/D) and Angiotensinogen (AGT) M235T (Li et al., 2001) of human RAAS system have been shown to be correlated with essential hypertension. The human ACE gene is located on chromosome 17q23, plays a substantial role in cardiovascular homeostasis (Lazartigues et al., 2007). A meta-analysis reported the association studies on ACE gene polymorphism and hypertension in 26 different populations (Agarwal et al., 2005). Recently, Srivastava et al. (2012b) have reported the #Kamna Srivastava is responsible for statistical design/analysis. E-mail: [email protected], [email protected] Address for correspondence: Dr. Kamna Srivastava, Molecular Cardiology Lab, Dr. B R Ambedkar Centre for Biomedical Research, University of Delhi, Delhi 110007, India. Tel: +91 11 27666272. Fax: +91 11 27666248. E-mail: [email protected]
History Received 3 February 2014 Revised 27 March 2014 Accepted 28 March 2014 Published online 9 May 2014
association of ACE I/D and AGT M235T (Srivastava et al., 2012a) gene polymorphism with essential hypertension in Indian context. Angiotensin-II production and cardiac ACE activity was shown to increase in hypertrophied rat hearts (Schunkert et al., 1990) and was linked with altered diastolic properties in hypertrophy during hypoperfusion (Eberli et al., 1992). Shiota et al. (1992) reported that the blood pressure and aortic ACE activity increases in the case of elevated levels of aorta ACE mRNA in animal model. To the best of our knowledge, the genetic expression of Angiotensin-converting enzyme in the whole blood of patients with essential hypertension has not been investigated so far. Hence, the purpose of the present study was to investigate the levels of Angiotensin-converting enzyme gene and protein expression in whole blood of patients with essential hypertension.
Materials and methods Study subjects An approval of Human ethics committee of All India Institute of Medical Sciences, New Delhi, was obtained prior to the study. The study was conducted in accordance with the guidelines of the Helsinki Declaration and written informed consent was obtained to participate in the study from each participants. Study subjects (52 Patients with essential hypertension and 42 matching controls), living in and around Delhi, and visiting the Department of Cardiology, All India Institute of Medical Sciences (AIIMS), New Delhi, were recruited on the basis of a standard questionnaire with inclusion and exclusion criteria. The study subjects were
Biomarkers Downloaded from informahealthcare.com by Memorial University of Newfoundland on 07/03/14 For personal use only.
DOI: 10.3109/1354750X.2014.910550
Angiotensin-converting enzyme gene in patients with essential hypertension
residents of northern India for past three generations of similar socio-economical-geographical background. The hypertensive patients in the age group 25–60 years, with B.P. 4140/90 mmHg were recruited while patients having secondary hypertension, smokers and alcoholics were excluded from the study. The controls were selected on the basis of age group 25–60 years, B.P. 5140/90 mmHg and absence of any antihypertensive treatment. Blood pressure was measured using Mercury sphygmomanometer and the patients were diagnosed according to the diagnostic standard of hypertension set by WHO/International Society on hypertension in 2005 and Joint National Committee-VII (JNC-VII) guidelines. The study subjects were seated in a chair with back support for at least 5 min prior to the measurement of blood pressure. The arm was placed comfortably on a table at heart level and feet were in flat position on the floor. The appropriate-sized cuff, based on the subject’s arm circumference was placed on the upper arm; lower edge of the cuff was 2–3 cm from the inner fold of arm. The blood pressure measurement was performed three times by the same doctor, with at least 1 min rest between each one. The antihypertensive medicines were withdrawn 24 h prior to the blood pressure measurement for patients with essential hypertension. Plasma lipid profile and blood glucose level were measured after overnight fasting in both patients and controls to rule out diabetes and hyperlipidaemia. The peripheral venous blood was drawn from the study subjects immediately after the blood pressure measurement. The results of the study were not used to influence the treatment given to the patient. Sample collection and processing The peripheral venous blood was withdrawn from the study subjects after 12 h of fasting and collected in the Ethylenediaminetetraacetate (EDTA) vial. About 1 ml of whole blood was used for RNA isolation and plasma was separated by centrifugation of the remaining volume of blood at 520 g for 10 min. The sample collection from patients and controls has been done within a span of 6-month time duration. The study for expression of protein from plasma was initiated within 1–2 h of sample collection. Isolation of RNA Total RNA was isolated from whole blood samples by QIAampÕ RNA Blood Mini Kit (QIAGENÕ ) according to the manufacturer’s instructions. Quantification of RNA was performed by Nanodrop (ND-1000). The yield of total RNA was in the range of 600–800 ng/ml of whole blood after extraction. Synthesis of cDNA Five hundred nanograms of total RNA was used for cDNA synthesis using First Strand cDNA Synthesis kit (Fermentas) according to the manufacturer’s protocol. Semi-quantitative reverse-trancription polymerase chain reaction analysis Semi-quantitative polymerase chain reaction (PCR) for ACE gene was performed by using Mastercycler gradient
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(eppendorf). Levels of ACE gene expression were quantified by densitometric analysis in terms of integrated densitometric value (IDV) by Image J software (NIH). The IDV obtained in study subjects was normalized to 18S rRNA gene expression. Real-time polymerase chain reaction analysis Real time quantitative PCR analysis for ACE gene expression was done by using ABI 7300 Real Time PCR System (Applied Biosystems, Foster City, CA) with SYBR green (Eurogentec) PCR Core reagents. Amplification was performed in triplicates in 15 ml volume, including 1 ml cDNA, 2.5 pmol of each primer in 2 Mesa green PCR Master mix (Eurogentec). To amplify human ACE transcript the following primers (Sigma, St. Louis, MO) based on NCBI reference sequence [GenBank: NM_000789.3] were used. Forward 50 -CCGAAATACGTGGAACTCATCAA-30 (exon 16, bases +2441 to +2463) Reverse 50 -CACGAGTCCCCTGCATCTACA-30 (exon 17, bases +2508 to +2488) 18S rRNA served as a housekeeping gene to access the overall cDNA content and the primers were as follows: Forward 50 -GTGGTGTTGAGGAAAGCAGACA-30 ; Reverse 50 -TGATCACACGTTCCACCTCATC-30 . After an initial holding step of 2 min at 50 C and 10 min at 95 C, samples were cycled 40 times at 95 C for 15 s and 60 C for 1 min. The specificity of assay was confirmed by melting curve analysis of PCR product. All assays were repeated thrice and Delta-Threshold cycle (DCT) values (the difference between the Threshold cycle (CT) values obtained for the gene of interest and normalizer or housekeeping gene), was calculated. The Delta-Delta-Threshold cycle (DDCT) equation was used to compare the expression of ACE gene in controls and patients in terms of fold difference according to the formula: DCT control ¼ [CT value of ACE gene in controlCT value of 18S rRNA gene in control]; DCT patient ¼ [CT value of ACE gene in patientCT value of 18S rRNA gene in patient] DDCT ¼ [Average DCT value of patientsAverage DCT value of controls] Fold difference ¼ [2]DDCT
Isolation of ACE protein and western blot Total protein was extracted from plasma by acetone precipitation method. Protein concentration was estimated by Bicinchoninic acid (BCA) protein estimation kit (Genei, Bangalore, India). Equal amount of proteins (50 mg) were separated on 8% SDS–polyacrylamide gels and transferred to PVDF membranes (MDI, India). The membranes were blocked overnight at 4 C with 3% Bovine Serum Albumin (BSA) and incubated for 3 h at room temperature with 1:1000 dilution of ACE primary antibody (H-170; sc-20791, Santa Cruz Biotechnology, Dallas, TX), followed by washing with Phosphate buffered saline (PBS) containing 0.5% Tween-20. Thereafter, the blots were incubated with horseradish peroxidase-conjugated secondary antibody of 1:5000 dilutions
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(sc-2357, Santa Cruz Biotechnology, Dallas, TX) and detected by chemiluminescence detection (WEST-ZOL Plus, Intron Biotechnology, Korea). Levels of ACE protein expression were quantified by densitometric analysis in terms of IDV. The protein expression was compared by the ratio of average IDV obtained for ACE protein in the study subjects to the IDV obtained for b-actin in the study subjects.
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Statistical analysis All statistical analysis was performed using GraphPad Prism version 5 (GraphPad Software Inc., San Diego, CA). Results are expressed as mean ± SD. The data were analyzed using unpaired t-test between the study groups. Correlation between blood pressures (systolic and diastolic) and IDV values were determined in both the study groups by Spearman’s test. Statistical significance is defined at a value of p50.05.
Results Base line characteristics of the study subjects
Biomarkers, 2014; 19(4): 314–318
Table 1. Baseline characteristics of the study subjects. Parameters Number (N) Sex (M/F) Age (years) BMI (kg/m2) Heart rate (beats/min) Blood glucose (mg/dl) Blood urea (mg/dl) Serum creatinine LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Total cholesterol (mg/dl) Triglyceride (mg/dl) Systolic blood pressure (SBP) mmHg Diastolic blood pressure (DBP) mmHg
Patients
Controls
p Value
52 31/21 49.8 ± 12 18.8 ± 3.4 76.2 ± 8.7 93.5 ± 17.6 21.8 ± 4.8 1.12 ± 0.3 89.3 ± 23.4 42.3 ± 7.2 159.3 ± 41.5 155.0 ± 44 150.0 ± 12.8
42 19/23 51.2 ± 6 18.08 ± 2.9 73.2 ± 6.2 88.7 ± 15.4 19.5 ± 4.2 0.92 ± 0.22 90.5 ± 29.4 39.8 ± 7.8 163.3 ± 37.5 149.0 ± 35 121 ± 3.8*
0.42 0.26 0.89 0.35 0.88 0.15 0.21 0.26 0.12 0.58 0.35 0.0001
97.2 ± 9.2
81.5 ± 3.5*
0.0001
BMI, body mass index; HDL, high-density lipoprotein; LDL, lowdensity lipoprotein. Patients group were compared with controls with t-test of significance or by chi-squared test. *p Value50.05 is considered to be significant.
Baseline parameters represent that the age between patients and controls were comparable and were non-smokers. Lipid profile values such as total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol were comparable in both the study subjects (Table 1). Systolic blood pressures (SBP) in patients were significantly higher (150.0 ± 12.8 mmHg) than controls (121 ± 3.8 mmHg). Similarly, diastolic blood pressures (DBP) in patients were higher (97.2 ± 9.2 mmHg) than controls (81.5 ± 3.5 mmHg). ACE gene expression Semi-quantitative analysis of ACE gene was determined by calculating the IDV through densitometry. The expression of ACE gene in terms of average IDV normalized to expression of 18S rRNA gene in patients and controls was 1.09 ± 0.12 and 0.83 ± 0.12, respectively (Figure 1). Semi-quantitative reverse transcription polymerase chain reaction indicated a small (1.3 times) but significant (p50.0001) increase in the ACE gene expression in patients than the controls. Quantitative relative expression of ACE gene was calculated according to housekeeping gene of 18S rRNA expression by DDCT method. All calculations were made according to median tests results of triplicate samples in both the study subjects separately. Relative gene expression in terms of average DCT value and fold difference of quantitative realtime PCR of patients and controls were calculated. The average DCT value for patients and controls were statistically significantly different (p50.001) between patients and controls (Table 2). The DDCT value for patients ( 4.8) shows the upregulated expression of ACE gene. The relative expression of mRNA for ACE gene to 18S rRNA in patients with essential hypertension was 28-fold higher as compared to control. ACE protein expression Figure 2 shows a representative western blot of plasma ACE and b-actin protein of patients with essential hypertension and controls. A significant difference for the ACE plasma protein
Figure 1. Semi-quantitative analysis of ACE gene expression. (A) Represents the semi-quantitative PCR of ACE gene and 18S rRNA (Housekeeping gene) in controls and patients, respectively. (B) Shows the relative ACE gene expression in terms of IDV of patients (1.09 ± 0.12) and normal healthy controls (0.83 ± 0.12). Results were expressed as densitometric ratio (ACE to 18S rRNA gene) in patients and controls group ± SD. *p50.0001. Table 2. Fold change expression and quantitative PCR results of ACE gene. Subjects Patients (N ¼ 52) Controls (N ¼ 42)
Average DCT
DDCT
Fold difference
4.57 ± 1.9* 9.38 ± 3.4*
4.8 0
28.1 1
CT, threshold cycle. 18S rRNA gene expression of the same samples was used for calculations. Fold change expression was calculated by DDCT method. *Data are means ± SD, p50.001.
expression level was observed in patients (1.9 ± 0.12) and control groups (0.72 ± 0.23, p ¼ 0.0001). The ACE protein level was found to be 2.7 times higher (approximately 164% increased IDV) in patients as compared to controls. The scatter plot between systolic blood pressure, diastolic blood pressure and ACE protein expression is given in Figure 3 to
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DOI: 10.3109/1354750X.2014.910550
Angiotensin-converting enzyme gene in patients with essential hypertension
Figure 2. Western blot analysis of ACE protein expression. (A) Representative western blots of ACE and b-actin protein in controls and patients, respectively. (B) Shows the relative ACE protein expression in terms of integrated densitometric value (IDV) of patients (1.9 ± 0.12) and normal healthy controls (0.72 ± 0.23). Results were expressed as densitometric ratio (ACE to b-actin protein) in patients and controls group ± SD. *p50.0001.
Figure 3. Scatter plot between systolic blood pressure (SBP), diastolic blood pressure (DBP) and ACE protein in study subjects. (A) Represents the ACE protein expression in terms of integrated densitometric value (IDV) with respect to systolic blood pressure (SBP) in study subjects. (B) Shows the ACE protein expression in terms of integrated densitometric value (IDV) with respect to diastolic blood pressure (DBP) in study subjects. Results were expressed as densitometric ratio (ACE to b-actin protein), SBP (mmHg) and DBP (mmHg) in study subjects.
show the distribution of individual data in both the study subjects. There was significant positive correlation found between systolic blood pressure ( ¼ 0.56, p ¼ 0.0001) and IDV values of protein in patients with essential hypertension. No such correlation ( ¼ 0.5, p ¼ 0.09) was found between systolic blood pressure and IDV values of protein in controls. Diastolic blood pressure and IDV values of protein in both patients ( ¼ 0.4, p ¼ 0.2) and controls ( ¼ 0.2, p ¼ 0.4) were also not correlated significantly.
Discussion Among the RAAS components, ACE1 catalyzes the cleavage of a dipeptide from the C-terminal end of angiotensin I leading to the formation of activated angiotensin II (Skeggs et al., 1956). ACE 2 plays a major role in antagonizing the actions of angiotensin II through its inactivation and
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formation of the agonist, Angiotensin 1–7. Increased level of ACE1 protein in spontaneously hypertensive rats is caused by the histone code modifications (Lee et al., 2012). Association of ACE2 with vascular changes is reported in case of hypertension and atherosclerosis (Heeneman et al., 2007). The renal ACE activity is involved in angiotensin II formation and plays a major role in the development of hypertension through the stimulation of sodium transport in the loop of Henle and the distal nephron (Gonzalez-Villalobos et al., 2011). Present study is a case–control study demonstrating the change in expression of ACE at mRNA and protein level in patients with essential hypertension. Shiota et al. (1992) have suggested an indispensible role of elevated ACE levels in increased angiotensin II formation resulting in hypertension in animal model. They further suggested the over-expression of ACE gene in inducing ACE biosynthesis in the vascular wall playing a major role in the development and management of hypertension in rats. Jankowski et al. (2011) suggested that angioprotectin, a vasoactive octapeptide has an antagonist role to the angiotensin II and is considered as new modulator of RAAS. Angiotensin A, a renal vasoconstrictor has binding affinity similar to angiotensin II in the context of hypertensive animal study (Yang et al., 2011). A newly discovered peptide of RAAS, Alamandine, has the similar cardio-protective actions as Angiotensin-(1-7) (Villela et al., 2014). Human ACE2 forms alamandine from angiotensin A, which has an important role in vasodilatation, central cardiovascular effects and antihypertensive effect in spontaneously hypertensive rats (Lautner et al., 2013). Other studies reported the regulatory role of ACE in the function of cardiovascular tissue via the determination of ACE activity (Grima et al., 1990; Hilgers et al., 1993; Okunishi et al., 1991) and measurement of conversion of Angiotensin-I to Angiotensin-II in stroke-prone spontaneously hypertensive rats (Hilgers et al., 1993). Okunishi et al. (1991) suggested that the elevated levels of vascular ACE activity are associated with the increase in systemic blood pressure in developmental phase of spontaneously hypertensive rats. However, most of these studies reported ACE expression in vascular tissue (Konoshita et al., 2006; Shiota et al., 1992). To the best of our knowledge, no study is available on ACE gene expression in at mRNA levels in human whole blood in patients with essential hypertension, worldwide. Comparing the levels of ACE gene expression in the peripheral blood of 52 patients with essential hypertension and 42 normal healthy volunteers as controls, by semiquantitative and quantitative Real Time PCR, and by western blot analysis clearly demonstrated an increase in relative expression of ACE mRNA and protein. In our study, the fold difference of circulating ACE gene expression in patients with essential hypertension was 11 times higher as compared to that of control group than that reported earlier for the aortic mRNA levels (2.6-fold) in early stage of hypertension (Shiota et al., 1992). This increase in mRNA for ACE gene also resulted in increased protein expression in the plasma (2.7fold increase) isolated from patient’s peripheral blood as compared to that of controls. This increase in protein expression was comparable with that reported earlier for ACE protein levels in aorta of CYP4A2-induced hypertensive
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rats (Sodhi et al., 2010). These findings clearly show that whole blood can be used for studying the levels of ACE mRNA and protein levels in patients with essential hypertension. Studies are in progress to study the role of ACE gene expression in the progression of disease, if any. The major limitation of our study is small sample size. Further studies in extended sample size are needed to understand the patho-physiological role of ACE gene expression in essential hypertension.
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Conclusions We have demonstrated that the expression of ACE gene and protein in whole blood cells of patients with essential hypertension was significantly higher as compared to the control group. Therefore, we suggest that the expression of ACE gene and protein is strongly involved with the regulation of blood pressure. However, a detail study in extended sample size with patients follow-up is warranted to confirm ACE gene and protein as prognostic biomarker.
Declaration of interest The author(s) declare that they have no conflicts of interests. This work was supported by Departmental grant, Dr. B R Ambedkar Centre for Biomedical Research, University of Delhi, India to Dr. Kamna Srivastava and Prof. Daman Saluja.
References Agarwal A, Williams GH, Fisher ND. (2005). Genetics of human hypertension. Trends Endocrinol Metab 16:127–33. Carey RM, Wang ZQ, Siragy HM. (2000). Role of the angiotensin type 2 receptor in the regulation of blood pressure and renal function. Hypertension 35:155–63. Carretero OA, Oparil S. (2000). Essential hypertension Part I: definition and etiology. Circulation 101:329–35. Eberli FR, Apstein CS, Ngoy S, Lorell BH. (1992). Exacerbation of left ventricular ischemic diastolic dysfunction by pressure overload hypertrophy: modification by specific inhibition of cardiac angiotensin converting enzyme. Circ Res 70:931–43. Gonzalez-Villalobos RA, Janjoulia T, Fletcher NK, et al. (2011). The absence of intrarenal ACE protects against hypertension. J Clin Invest 123:2011–23. Grima M, Welsch C, Giesen-Crouse EM, et al. (1990). Age-related variations in tissue angiotensin converting enzyme activities: comparison between spontaneously hypertensive and Wistar-Kyoto rats. J Hypertens 8:697–702. Hall JE, Guyton AC. (2006). Textbook of medical physiology. 11th ed. St. Louis: Elsevier Saunders, 228. Heeneman S, Sluimer JC, Daemen MJ. (2007). Angiotensin-converting enzyme and vascular remodeling. Circ Res 101:441–54.
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Hilgers KF, Veelken R, Mai M, et al. (1993). Vascular conversion of angiotensin I in stroke-prone spontaneously hypertensive and WistarKyoto rats. J Hypertens 11:1053–9. Jankowski V, To¨lle M, Santos RA, et al. (2011). Angioprotectin: an angiotensin II-like peptide causing vasodilatory effects. FASEB J 25: 2987–95. Konoshita T, Wakahara S, Mizuno S, et al. (2006). Tissue gene expression of renin-angiotensin system in human type 2 diabetic nephropathy. Diabetes Care 29:848–52. Lautner RQ, Villela DC, Fraga-Silva RA, et al. (2013). Discovery and characterization of alamandine: a novel component of the reninangiotensin system. Circ Res 112:1104–11. Lazartigues E, Feng Y, Lavoie JL. (2007). The two fACEs of the tissue renin-angiotensin systems: implication in cardiovascular diseases. Curr Pharm Des 13:1231–45. Lee HA, Cho HM, Lee DY, et al. (2012). Tissue-specific upregulation of angiotensin-converting enzyme 1 in spontaneously hypertensive rats through histone code modifications. Hypertension 59:621–6. Li X, Wang L, Han X. (2001). Association between angiotensin system gene polymorphism and essential hypertension. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 18:292–5. Okunishi H, Kawamoto T, Kurobe Y, et al. (1991). Pathogenetic role of vascular angiotensin-converting enzyme in the spontaneously hypertensive rat. Clin Exp Pharmacol Physiol 18:649–59. Oparil S, Zaman MA, Calhoun DA. (2003). Pathogenesis of hypertension. Ann Intern Med 139:761–76. Schunkert H, Dzau VJ, Tang SS, et al. (1990). Increased rat cardiac angiotensin converting enzyme activity and mRNA expression in pressure overload left ventricular hypertrophy. Effects on resistance, contractility, and relaxation. J Clin Invest 86:1913–20. Shiota N, Miyazaki M, Okunishi H. (1992). Increase of angiotensin converting enzyme gene expression in the hypertensive aorta. Hypertension 20:168–74. Skeggs Jr. LT, Kahn JR, Shumway NP. (1956). The preparation and function of the hypertensin-converting enzyme. J Exp Med 103: 295–9. Sodhi K, Wu CC, Cheng J, et al. (2010). CYP4A2-induced hypertension is 20-hydroxyeicosatetraenoic acid- and angiotensin II-dependent. Hypertension 56:871–8. Srivastava K, Chandra S, Bhatia J, et al. (2012a). Association of angiotensinogen (M235T) gene polymorphism with blood pressure lowering response to angiotensin converting enzyme inhibitor (Enalapril). J Pharm Pharm Sci 15:399–406. Srivastava K, Sundriyal R, Meena PC, et al. (2012b). Association of angiotensin converting enzyme (insertion/deletion) gene polymorphism with essential hypertension in Northern Indian subjects. Genet Test Mol Biomarkers 16:174–7. Villela DC, Passos-Silva DG, Santos RA. (2014). Alamandine: a new member of the angiotensin family. Curr Opin Nephrol Hypertens 23: 130–4. Wang JL, Li Xue, Hao PP, et al. (2010). Angiotensin II type 1 receptor gene A1166C polymorphism and essential hypertension in Chinese a meta-analysis. J Renin-Angiotensin-Aldosterone Syst 11:127–35. Yang R, Smolders I, Vanderheyden P, et al. (2011). Pressor and renal hemodynamic effects of the novel angiotensin A peptide are angiotensin II type 1A receptor dependent. Hypertension 57: 956–64.
RESEARCH ARTICLE
Expression of angiotensin-converting enzyme gene in whole blood in patients with essential hypertension Sudhir Chandra1, Rajiv Narang2, Daman Saluja1, Jagriti Bhatia3, and Kamna Srivastava1#
Biomarkers Downloaded from informahealthcare.com by Memorial University of Newfoundland on 07/03/14 For personal use only.
1
Dr. B R Ambedkar Centre for Biomedical Research, University of Delhi, Delhi, India, 2Department of Cardiology, and 3Department of Pharmacology, All India Institute of Medical Sciences, New Delhi, India Abstract
Keywords
Objective: The present study aims to investigate the correlation of the angiotensin-converting enzyme (ACE) gene expression and protein expression in patients with essential hypertension in whole blood. Methods: ACE gene expression was analyzed by Real Time PCR and western blot in 52 patients with essential hypertension and 42 healthy controls. Results: We observed a significant increase in Delta threshold cycle (DCT) values in the circulating ACE gene and ACE protein expression in patients as compared to controls. Conclusions: The up-regulation in relative expression of circulating Angiotensin converting enzyme mRNA and protein in patients with respect to controls might be correlated with high blood pressure in patients with essential hypertension.
Angiotensin-converting enzyme, blood pressure, essential hypertension, gene expression, renin–angiotensin–aldosterone system
Introduction Hypertension is major public health problem all over the world, including India. Essential hypertension is the form of hypertension that has no identifiable cause but it affects 95% of hypertensive patients (Carretero & Oparil, 2000; Hall & Guyton, 2006; Oparil et al., 2003) probably due to the consequence of an interaction between environmental and genetic factors. Angiotensin-converting enzyme (ACE), an important component of renin–angiotensin–aldosterone system (RAAS) converts angiotensin I into a physiologically active peptide angiotensin II, a potent vasopressor and aldosteronestimulating peptide that controls blood pressure and fluidelectrolyte balance (Carey et al., 2000). Several genes of RAAS such as Angiotensin II type 1 receptor (AT1R) (Wang et al., 2010), ACE insertion/deletion (ACE I/D) and Angiotensinogen (AGT) M235T (Li et al., 2001) of human RAAS system have been shown to be correlated with essential hypertension. The human ACE gene is located on chromosome 17q23, plays a substantial role in cardiovascular homeostasis (Lazartigues et al., 2007). A meta-analysis reported the association studies on ACE gene polymorphism and hypertension in 26 different populations (Agarwal et al., 2005). Recently, Srivastava et al. (2012b) have reported the #Kamna Srivastava is responsible for statistical design/analysis. E-mail: [email protected], [email protected] Address for correspondence: Dr. Kamna Srivastava, Molecular Cardiology Lab, Dr. B R Ambedkar Centre for Biomedical Research, University of Delhi, Delhi 110007, India. Tel: +91 11 27666272. Fax: +91 11 27666248. E-mail: [email protected]
History Received 3 February 2014 Revised 27 March 2014 Accepted 28 March 2014 Published online 9 May 2014
association of ACE I/D and AGT M235T (Srivastava et al., 2012a) gene polymorphism with essential hypertension in Indian context. Angiotensin-II production and cardiac ACE activity was shown to increase in hypertrophied rat hearts (Schunkert et al., 1990) and was linked with altered diastolic properties in hypertrophy during hypoperfusion (Eberli et al., 1992). Shiota et al. (1992) reported that the blood pressure and aortic ACE activity increases in the case of elevated levels of aorta ACE mRNA in animal model. To the best of our knowledge, the genetic expression of Angiotensin-converting enzyme in the whole blood of patients with essential hypertension has not been investigated so far. Hence, the purpose of the present study was to investigate the levels of Angiotensin-converting enzyme gene and protein expression in whole blood of patients with essential hypertension.
Materials and methods Study subjects An approval of Human ethics committee of All India Institute of Medical Sciences, New Delhi, was obtained prior to the study. The study was conducted in accordance with the guidelines of the Helsinki Declaration and written informed consent was obtained to participate in the study from each participants. Study subjects (52 Patients with essential hypertension and 42 matching controls), living in and around Delhi, and visiting the Department of Cardiology, All India Institute of Medical Sciences (AIIMS), New Delhi, were recruited on the basis of a standard questionnaire with inclusion and exclusion criteria. The study subjects were
Biomarkers Downloaded from informahealthcare.com by Memorial University of Newfoundland on 07/03/14 For personal use only.
DOI: 10.3109/1354750X.2014.910550
Angiotensin-converting enzyme gene in patients with essential hypertension
residents of northern India for past three generations of similar socio-economical-geographical background. The hypertensive patients in the age group 25–60 years, with B.P. 4140/90 mmHg were recruited while patients having secondary hypertension, smokers and alcoholics were excluded from the study. The controls were selected on the basis of age group 25–60 years, B.P. 5140/90 mmHg and absence of any antihypertensive treatment. Blood pressure was measured using Mercury sphygmomanometer and the patients were diagnosed according to the diagnostic standard of hypertension set by WHO/International Society on hypertension in 2005 and Joint National Committee-VII (JNC-VII) guidelines. The study subjects were seated in a chair with back support for at least 5 min prior to the measurement of blood pressure. The arm was placed comfortably on a table at heart level and feet were in flat position on the floor. The appropriate-sized cuff, based on the subject’s arm circumference was placed on the upper arm; lower edge of the cuff was 2–3 cm from the inner fold of arm. The blood pressure measurement was performed three times by the same doctor, with at least 1 min rest between each one. The antihypertensive medicines were withdrawn 24 h prior to the blood pressure measurement for patients with essential hypertension. Plasma lipid profile and blood glucose level were measured after overnight fasting in both patients and controls to rule out diabetes and hyperlipidaemia. The peripheral venous blood was drawn from the study subjects immediately after the blood pressure measurement. The results of the study were not used to influence the treatment given to the patient. Sample collection and processing The peripheral venous blood was withdrawn from the study subjects after 12 h of fasting and collected in the Ethylenediaminetetraacetate (EDTA) vial. About 1 ml of whole blood was used for RNA isolation and plasma was separated by centrifugation of the remaining volume of blood at 520 g for 10 min. The sample collection from patients and controls has been done within a span of 6-month time duration. The study for expression of protein from plasma was initiated within 1–2 h of sample collection. Isolation of RNA Total RNA was isolated from whole blood samples by QIAampÕ RNA Blood Mini Kit (QIAGENÕ ) according to the manufacturer’s instructions. Quantification of RNA was performed by Nanodrop (ND-1000). The yield of total RNA was in the range of 600–800 ng/ml of whole blood after extraction. Synthesis of cDNA Five hundred nanograms of total RNA was used for cDNA synthesis using First Strand cDNA Synthesis kit (Fermentas) according to the manufacturer’s protocol. Semi-quantitative reverse-trancription polymerase chain reaction analysis Semi-quantitative polymerase chain reaction (PCR) for ACE gene was performed by using Mastercycler gradient
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(eppendorf). Levels of ACE gene expression were quantified by densitometric analysis in terms of integrated densitometric value (IDV) by Image J software (NIH). The IDV obtained in study subjects was normalized to 18S rRNA gene expression. Real-time polymerase chain reaction analysis Real time quantitative PCR analysis for ACE gene expression was done by using ABI 7300 Real Time PCR System (Applied Biosystems, Foster City, CA) with SYBR green (Eurogentec) PCR Core reagents. Amplification was performed in triplicates in 15 ml volume, including 1 ml cDNA, 2.5 pmol of each primer in 2 Mesa green PCR Master mix (Eurogentec). To amplify human ACE transcript the following primers (Sigma, St. Louis, MO) based on NCBI reference sequence [GenBank: NM_000789.3] were used. Forward 50 -CCGAAATACGTGGAACTCATCAA-30 (exon 16, bases +2441 to +2463) Reverse 50 -CACGAGTCCCCTGCATCTACA-30 (exon 17, bases +2508 to +2488) 18S rRNA served as a housekeeping gene to access the overall cDNA content and the primers were as follows: Forward 50 -GTGGTGTTGAGGAAAGCAGACA-30 ; Reverse 50 -TGATCACACGTTCCACCTCATC-30 . After an initial holding step of 2 min at 50 C and 10 min at 95 C, samples were cycled 40 times at 95 C for 15 s and 60 C for 1 min. The specificity of assay was confirmed by melting curve analysis of PCR product. All assays were repeated thrice and Delta-Threshold cycle (DCT) values (the difference between the Threshold cycle (CT) values obtained for the gene of interest and normalizer or housekeeping gene), was calculated. The Delta-Delta-Threshold cycle (DDCT) equation was used to compare the expression of ACE gene in controls and patients in terms of fold difference according to the formula: DCT control ¼ [CT value of ACE gene in controlCT value of 18S rRNA gene in control]; DCT patient ¼ [CT value of ACE gene in patientCT value of 18S rRNA gene in patient] DDCT ¼ [Average DCT value of patientsAverage DCT value of controls] Fold difference ¼ [2]DDCT
Isolation of ACE protein and western blot Total protein was extracted from plasma by acetone precipitation method. Protein concentration was estimated by Bicinchoninic acid (BCA) protein estimation kit (Genei, Bangalore, India). Equal amount of proteins (50 mg) were separated on 8% SDS–polyacrylamide gels and transferred to PVDF membranes (MDI, India). The membranes were blocked overnight at 4 C with 3% Bovine Serum Albumin (BSA) and incubated for 3 h at room temperature with 1:1000 dilution of ACE primary antibody (H-170; sc-20791, Santa Cruz Biotechnology, Dallas, TX), followed by washing with Phosphate buffered saline (PBS) containing 0.5% Tween-20. Thereafter, the blots were incubated with horseradish peroxidase-conjugated secondary antibody of 1:5000 dilutions
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(sc-2357, Santa Cruz Biotechnology, Dallas, TX) and detected by chemiluminescence detection (WEST-ZOL Plus, Intron Biotechnology, Korea). Levels of ACE protein expression were quantified by densitometric analysis in terms of IDV. The protein expression was compared by the ratio of average IDV obtained for ACE protein in the study subjects to the IDV obtained for b-actin in the study subjects.
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Statistical analysis All statistical analysis was performed using GraphPad Prism version 5 (GraphPad Software Inc., San Diego, CA). Results are expressed as mean ± SD. The data were analyzed using unpaired t-test between the study groups. Correlation between blood pressures (systolic and diastolic) and IDV values were determined in both the study groups by Spearman’s test. Statistical significance is defined at a value of p50.05.
Results Base line characteristics of the study subjects
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Table 1. Baseline characteristics of the study subjects. Parameters Number (N) Sex (M/F) Age (years) BMI (kg/m2) Heart rate (beats/min) Blood glucose (mg/dl) Blood urea (mg/dl) Serum creatinine LDL cholesterol (mg/dl) HDL cholesterol (mg/dl) Total cholesterol (mg/dl) Triglyceride (mg/dl) Systolic blood pressure (SBP) mmHg Diastolic blood pressure (DBP) mmHg
Patients
Controls
p Value
52 31/21 49.8 ± 12 18.8 ± 3.4 76.2 ± 8.7 93.5 ± 17.6 21.8 ± 4.8 1.12 ± 0.3 89.3 ± 23.4 42.3 ± 7.2 159.3 ± 41.5 155.0 ± 44 150.0 ± 12.8
42 19/23 51.2 ± 6 18.08 ± 2.9 73.2 ± 6.2 88.7 ± 15.4 19.5 ± 4.2 0.92 ± 0.22 90.5 ± 29.4 39.8 ± 7.8 163.3 ± 37.5 149.0 ± 35 121 ± 3.8*
0.42 0.26 0.89 0.35 0.88 0.15 0.21 0.26 0.12 0.58 0.35 0.0001
97.2 ± 9.2
81.5 ± 3.5*
0.0001
BMI, body mass index; HDL, high-density lipoprotein; LDL, lowdensity lipoprotein. Patients group were compared with controls with t-test of significance or by chi-squared test. *p Value50.05 is considered to be significant.
Baseline parameters represent that the age between patients and controls were comparable and were non-smokers. Lipid profile values such as total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol were comparable in both the study subjects (Table 1). Systolic blood pressures (SBP) in patients were significantly higher (150.0 ± 12.8 mmHg) than controls (121 ± 3.8 mmHg). Similarly, diastolic blood pressures (DBP) in patients were higher (97.2 ± 9.2 mmHg) than controls (81.5 ± 3.5 mmHg). ACE gene expression Semi-quantitative analysis of ACE gene was determined by calculating the IDV through densitometry. The expression of ACE gene in terms of average IDV normalized to expression of 18S rRNA gene in patients and controls was 1.09 ± 0.12 and 0.83 ± 0.12, respectively (Figure 1). Semi-quantitative reverse transcription polymerase chain reaction indicated a small (1.3 times) but significant (p50.0001) increase in the ACE gene expression in patients than the controls. Quantitative relative expression of ACE gene was calculated according to housekeeping gene of 18S rRNA expression by DDCT method. All calculations were made according to median tests results of triplicate samples in both the study subjects separately. Relative gene expression in terms of average DCT value and fold difference of quantitative realtime PCR of patients and controls were calculated. The average DCT value for patients and controls were statistically significantly different (p50.001) between patients and controls (Table 2). The DDCT value for patients ( 4.8) shows the upregulated expression of ACE gene. The relative expression of mRNA for ACE gene to 18S rRNA in patients with essential hypertension was 28-fold higher as compared to control. ACE protein expression Figure 2 shows a representative western blot of plasma ACE and b-actin protein of patients with essential hypertension and controls. A significant difference for the ACE plasma protein
Figure 1. Semi-quantitative analysis of ACE gene expression. (A) Represents the semi-quantitative PCR of ACE gene and 18S rRNA (Housekeeping gene) in controls and patients, respectively. (B) Shows the relative ACE gene expression in terms of IDV of patients (1.09 ± 0.12) and normal healthy controls (0.83 ± 0.12). Results were expressed as densitometric ratio (ACE to 18S rRNA gene) in patients and controls group ± SD. *p50.0001. Table 2. Fold change expression and quantitative PCR results of ACE gene. Subjects Patients (N ¼ 52) Controls (N ¼ 42)
Average DCT
DDCT
Fold difference
4.57 ± 1.9* 9.38 ± 3.4*
4.8 0
28.1 1
CT, threshold cycle. 18S rRNA gene expression of the same samples was used for calculations. Fold change expression was calculated by DDCT method. *Data are means ± SD, p50.001.
expression level was observed in patients (1.9 ± 0.12) and control groups (0.72 ± 0.23, p ¼ 0.0001). The ACE protein level was found to be 2.7 times higher (approximately 164% increased IDV) in patients as compared to controls. The scatter plot between systolic blood pressure, diastolic blood pressure and ACE protein expression is given in Figure 3 to
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DOI: 10.3109/1354750X.2014.910550
Angiotensin-converting enzyme gene in patients with essential hypertension
Figure 2. Western blot analysis of ACE protein expression. (A) Representative western blots of ACE and b-actin protein in controls and patients, respectively. (B) Shows the relative ACE protein expression in terms of integrated densitometric value (IDV) of patients (1.9 ± 0.12) and normal healthy controls (0.72 ± 0.23). Results were expressed as densitometric ratio (ACE to b-actin protein) in patients and controls group ± SD. *p50.0001.
Figure 3. Scatter plot between systolic blood pressure (SBP), diastolic blood pressure (DBP) and ACE protein in study subjects. (A) Represents the ACE protein expression in terms of integrated densitometric value (IDV) with respect to systolic blood pressure (SBP) in study subjects. (B) Shows the ACE protein expression in terms of integrated densitometric value (IDV) with respect to diastolic blood pressure (DBP) in study subjects. Results were expressed as densitometric ratio (ACE to b-actin protein), SBP (mmHg) and DBP (mmHg) in study subjects.
show the distribution of individual data in both the study subjects. There was significant positive correlation found between systolic blood pressure ( ¼ 0.56, p ¼ 0.0001) and IDV values of protein in patients with essential hypertension. No such correlation ( ¼ 0.5, p ¼ 0.09) was found between systolic blood pressure and IDV values of protein in controls. Diastolic blood pressure and IDV values of protein in both patients ( ¼ 0.4, p ¼ 0.2) and controls ( ¼ 0.2, p ¼ 0.4) were also not correlated significantly.
Discussion Among the RAAS components, ACE1 catalyzes the cleavage of a dipeptide from the C-terminal end of angiotensin I leading to the formation of activated angiotensin II (Skeggs et al., 1956). ACE 2 plays a major role in antagonizing the actions of angiotensin II through its inactivation and
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formation of the agonist, Angiotensin 1–7. Increased level of ACE1 protein in spontaneously hypertensive rats is caused by the histone code modifications (Lee et al., 2012). Association of ACE2 with vascular changes is reported in case of hypertension and atherosclerosis (Heeneman et al., 2007). The renal ACE activity is involved in angiotensin II formation and plays a major role in the development of hypertension through the stimulation of sodium transport in the loop of Henle and the distal nephron (Gonzalez-Villalobos et al., 2011). Present study is a case–control study demonstrating the change in expression of ACE at mRNA and protein level in patients with essential hypertension. Shiota et al. (1992) have suggested an indispensible role of elevated ACE levels in increased angiotensin II formation resulting in hypertension in animal model. They further suggested the over-expression of ACE gene in inducing ACE biosynthesis in the vascular wall playing a major role in the development and management of hypertension in rats. Jankowski et al. (2011) suggested that angioprotectin, a vasoactive octapeptide has an antagonist role to the angiotensin II and is considered as new modulator of RAAS. Angiotensin A, a renal vasoconstrictor has binding affinity similar to angiotensin II in the context of hypertensive animal study (Yang et al., 2011). A newly discovered peptide of RAAS, Alamandine, has the similar cardio-protective actions as Angiotensin-(1-7) (Villela et al., 2014). Human ACE2 forms alamandine from angiotensin A, which has an important role in vasodilatation, central cardiovascular effects and antihypertensive effect in spontaneously hypertensive rats (Lautner et al., 2013). Other studies reported the regulatory role of ACE in the function of cardiovascular tissue via the determination of ACE activity (Grima et al., 1990; Hilgers et al., 1993; Okunishi et al., 1991) and measurement of conversion of Angiotensin-I to Angiotensin-II in stroke-prone spontaneously hypertensive rats (Hilgers et al., 1993). Okunishi et al. (1991) suggested that the elevated levels of vascular ACE activity are associated with the increase in systemic blood pressure in developmental phase of spontaneously hypertensive rats. However, most of these studies reported ACE expression in vascular tissue (Konoshita et al., 2006; Shiota et al., 1992). To the best of our knowledge, no study is available on ACE gene expression in at mRNA levels in human whole blood in patients with essential hypertension, worldwide. Comparing the levels of ACE gene expression in the peripheral blood of 52 patients with essential hypertension and 42 normal healthy volunteers as controls, by semiquantitative and quantitative Real Time PCR, and by western blot analysis clearly demonstrated an increase in relative expression of ACE mRNA and protein. In our study, the fold difference of circulating ACE gene expression in patients with essential hypertension was 11 times higher as compared to that of control group than that reported earlier for the aortic mRNA levels (2.6-fold) in early stage of hypertension (Shiota et al., 1992). This increase in mRNA for ACE gene also resulted in increased protein expression in the plasma (2.7fold increase) isolated from patient’s peripheral blood as compared to that of controls. This increase in protein expression was comparable with that reported earlier for ACE protein levels in aorta of CYP4A2-induced hypertensive
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rats (Sodhi et al., 2010). These findings clearly show that whole blood can be used for studying the levels of ACE mRNA and protein levels in patients with essential hypertension. Studies are in progress to study the role of ACE gene expression in the progression of disease, if any. The major limitation of our study is small sample size. Further studies in extended sample size are needed to understand the patho-physiological role of ACE gene expression in essential hypertension.
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Conclusions We have demonstrated that the expression of ACE gene and protein in whole blood cells of patients with essential hypertension was significantly higher as compared to the control group. Therefore, we suggest that the expression of ACE gene and protein is strongly involved with the regulation of blood pressure. However, a detail study in extended sample size with patients follow-up is warranted to confirm ACE gene and protein as prognostic biomarker.
Declaration of interest The author(s) declare that they have no conflicts of interests. This work was supported by Departmental grant, Dr. B R Ambedkar Centre for Biomedical Research, University of Delhi, India to Dr. Kamna Srivastava and Prof. Daman Saluja.
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