http://informahealthcare.com/bmk ISSN: 1354-750X (print), 1366-5804 (electronic) Biomarkers, 2013; 18(8): 716–720 ! 2013 Informa UK Ltd. DOI: 10.3109/1354750X.2013.847122
RESEARCH ARTICLE
Cyclophilin-A: a novel biomarker for untreated male essential hypertension Chen-Shu Chang1,2, Shih-Li Su3,4, Chia-Chu Chang5, Kwo-Whei Lee6, Chen-Ling Kuo7, Ching-Shan Huang7, Wan-Min Tseng7, and Chin-San Liu1,7,8 Department of Neurology, Changhua Christian Hospital, Changhua, Taiwan, 2Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science of Technology, Taichung, Taiwan, 3Division of Endocrinology and Metabolism, Department of Internal Medicine, Diabetes Education Center, Changhua Christian Hospital, Changhua, Taiwan, 4Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, 5Division of Nephrology, Department of Internal Medicine, 6Department of Medical Imaging, 7Vascular and Genomic Research Center, Changhua Christian Hospital, Changhua, Taiwan, and 8Graduate Institute of Integrative Medicine, China Medical University, Taichung, Taiwan Abstract
Keywords
Vascular cytokines, total nitrite, and cyclophilin-A (CyP-A) may be related to the pathogenesis of untreated hypertension. Forty males with normotensive and untreated essential hypertension were recruited in this cytokines survey. Body mass index (BMI), hyperlipidemia, and plasma CyP-A were increased in the hypertensive group (p50.05). However, only BMI (p ¼ 0.022) and plasma CyP-A (p ¼ 0.020) were found to be significant contributors to hypertension by multiple regression analysis. CyP-A was also positively correlated with systolic blood pressure (p ¼ 0.029) and diastolic blood pressure (p ¼ 0.047). These findings indicated that plasma CyP-A is a critical molecular biomarker in the early pathogenesis of essential hypertension.
Atherosclerosis, cyclophilin-A, cytokine, hypertension
Introduction The pathogenesis of arterial hypertension is multifactorial (Oparil et al., 2003). Major physiological alterations have been identified in hypertensive individuals, including vascular hypertrophy (Olsen et al., 2002), systemic inflammation (Bermudez et al., 2002), endothelial dysfunction (Iiyama et al., 1996), and enhanced oxidative stress (Lacy et al., 1998). These mechanisms ordinarily occur in an insidious and asymptomatic pattern over the development course of hypertension. Several cytokines have been found to be involved in the pathogenesis of hypertension-related atherosclerosis. Vascular endothelial function can be assessed by matrix metalloproteinase (MMP) in many cardiovascular diseases including hypertension and its chronic complications (Lim et al., 2010). Increased vascular expressions of MMP-2 and MMP-9 were found in hypertensive subjects (Li-Saw-Hee et al., 2000; Onal et al., 2009), but another study reported decreased MMP-2 and MMP-9 in hypertensive individuals (Panek & Bader, 2006). Biomarkers of endothelial cell activation are generally surveyed in hypertension, including adhesion molecule soluble endothelium-selectin (sE-selectin) (Blann et al., 1994), soluble intercellular adhesion molecule-1 Address for correspondence: Chin-San Liu, MD, PhD, Department of Neurology, Vascular and Genomic Research Center, Changhua Christian Hospital, 135 Nanhsiao Street, Changhua 500, Taiwan. Tel: +886-47238595 ext.4752. Fax: +886-4-7238595 ext.4063. E-mail: liu48111@ gmail.com
History Received 13 August 2013 Revised 13 September 2013 Accepted 17 September 2013 Published online 28 October 2013
(sICAM-1) (Komatsu et al., 1997), and soluble vascular cell adhesion molecule-1 (sVCAM-1) (Kuroda et al., 2001), but there are inconsistencies in the published literature (Hwang et al., 1997; Miller et al., 2004). Elevation of the concentrations of plasma interleukin-2 (IL-2), interleukin-4 (IL-4) (Tuttle & Boppana, 1990) and total nitrite/nitrate concentrations (NOx) (Touyz, 2000) was found in several basic and clinical studies of hypertension, but there some disputed and controversial results (Davey Smith et al., 2005; Kato et al., 1999). Cyclophilin-A (CyP-A), the most abundant form of cyclophilin in human intracellular protein, is secreted by monocytes/macrophages, endothelial cells, and vascular smooth muscle cells in inflammatory and oxidative stimuli in arterial atherosclerosis (Jin et al., 2000; Satoh et al., 2008). CyP-A can elicit leukocyte chemotaxis with raised levels in inflammatory tissues in vitro, was demonstrated to promote atherosclerosis by increasing low-density lipoprotein (LDL) uptake and stimulating recruitment of inflammatory cells in the vessel wall with an oxidative stress environment of the Apoe/Ppia þ/þ mice (Nigro et al., 2011). In the present study, CyP-A was also recruited for potential cytokine screening as a biomarker of hypertension due to its characteristic of inflammatory responses in vascular system (Arora et al., 2005; Handschumacher et al., 1984; Jin et al., 2000; Satoh et al., 2008). The above-mentioned cytokines are all possibly involved in the pathogenesis of essential hypertension during the initial stage. Thus, cytokine biomarkers in response to inflammation-related atherosclerosis are of
20 13
Biomarkers Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
1
DOI: 10.3109/1354750X.2013.847122
interest in the evaluation of the early stage of essential hypertension before a symptomatic event. Therefore, our hypothesis is that alterations of the plasma levels of MMP-2, MMP-9, sE-selectin, sICAM, sVCAM, IL-2, IL-4, NOx, and CyP-A may occur in the early pathogenesis of essential hypertension.
CyP-A for untreated male essential hypertension
717
dihydrochloride) as described by Green et al. (1982). The assay requires conversion of nitrate to nitrite using nitrate reductase, then Griess reagent is added, which converts nitrite into a deep-purple azo compound. Absorption measurements were carried out at 540 nm using a microplate reader. Biochemical measurements
Methods
Biomarkers Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
Study subjects Forty healthy non-hypertensive males and 40 age-matched untreated males with essential hypertension were recruited from the health clinic of Changhua Christian Hospital from September 2009 to October, 2010. The untreated essential hypertension group was defined as those without medication with an average home systolic blood pressure greater than 140 mmHg and a diastolic blood pressure greater than 90 mmHg according to the definition of stage I/II hypertension in the JNC-7 guidelines. All subjects were in good health and free of any cardiovascular diseases as assessed by their medical history, physical examination and routine screening laboratory data taken by an internist and family doctors. Subjects without alcoholism and diabetes (fasting blood glucose less than 110 mg/dL), except for some with mild hyperlipidemia, were recruited for the study. Subjects actively taking medication were excluded. The smoking history of the study subjects was obtained by questionnaire. Passive smoking exposure was not considered in this study, and our nonsmokers had no previous smoking experience. All of the human experimental procedures followed the ethical standards of Changhua Christian Hospital and were approved by the institutional review committee. Informed consent for study enrollment was obtained from each of the subjects. Blood samples were taken from each of the subjects in the morning after he or she had fasted overnight and before smoking if he or she was a smoker. Ten milliliters of whole blood were withdrawn from an antecubital vein. Plasma and serum were immediately collected by centrifugation, divided into several aliquots, and stored at 80 C until analysis. Plasma cytokines measurement The quality of samples in 80 C refrigerator was stable after storage of recruited whole blood within 5 months. Three samples were repeatedly measured in the estimation of all cytokines and lipid profiles at 1st day and 3rd month of samples collection. No statistic difference was observed in this pilot calibration study in the same sample after repeated measurement with 3 months. All biochemical studies of samples would be performed with 3 months. Plasma CyP-A was measured using commercially-available enzyme-linked immunosorbent assay kits (Wuhan USCN Science, Wuhan, China) in accordance with the manufacturer’s instructions. Plasma total MMP-2 and MMP-9 levels were measured by ELISA (R&D Systems, Minneapolis, MN). Plasma IL-2 and IL-4 levels were measured by ELISA (eBioscience, San Diego, CA) according to the manufacturer’s instructions. Plasma sVCAM-1 and sE-selectin levels were measured by ELISA (R&D Systems). The plasma NOx level was measured by Griess reagent (0.1% N-(1-naphthyl) ethylenediamine
Total serum cholesterol was determined enzymatically using the Sigma Diagnostics cholesterol reagent (Sigma Chemical Co., St. Louis, MO). Serum high density lipoprotein cholesterol (HDL-C) was determined using the Sigma Diagnostics HDL-C reagent (Warnick et al., 1982). Dextran sulfate and magnesium ions were used for precipitation and isolation of LDL and very LDL particles. Serum triglyceride concentrations were determined using the Sigma Diagnostics triglyceride reagent following the modification described by McGowan et al. (1983). LDL-C concentration was calculated according to the formula developed by Friedewald et al. (1972). Statistical analysis Data are expressed as the mean SD. All statistical analyses were performed using the SPSS statistical package, version 17.0 (Chicago, IL). The significances of the differences between the mean values in the non-hypertension and hypertension groups were determined by paired t-test and one-way analysis of variance (ANOVA) under control of BMI, smoking index, and lipid profile. Multiple linear regression was applied to assess the importance of various contributing factors to hypertension. Partial correlation was applied in the comparison between blood pressure and the plasma level of CyP-A under control of BMI, smoking index, and lipid profile.
Results The baseline demographic data of the 40 hypertensive and 40 non-hypertensive gentlemen enrolled in this study are shown in Table 1. The hypertension group revealed a higher body mass index (BMI) than the non-hypertension group (25.1 2.3 versus 23.0 2.8; p ¼ 0.001). The hypertension group had higher cholesterol (207.0 35.0 versus 193.5 36.7, p ¼ 0.036), LDL-C (126.0 31.0 versus 113.4 32.9, p ¼ 0.028) and triglyceride (132.0 60.3 versus 97.5 57.3, p ¼ 0.001) than the non-hypertension group. In addition, HDL-C (54.6 61.2 versus 61.2 15.1, p ¼ 0.005) was decreased in the non-hypertension group. Additionally, plasma CyP-A was higher in the hypertension group than in the non-hypertension group (94.5 26.2 versus 73.1 29.8; p ¼ 0.001). However, blood pressure had no effect on the other biochemical data, including MMP-2, MMP-9, adhesion molecules, IL-2, IL-4, and nitric oxide (NOx). To assess the significant factors contributing to hypertension shown in Table 1, multiple linear regression analysis was applied, and the results are presented in Table 2. We found that smoking index, cholesterol, LDL-C, HDL-C, and triglyceride level did not contribute significantly to hypertension, except BMI (p ¼ 0.022) and plasma CyP-A (p ¼ 0.020). The increased CyP-A concentration in the study
718
C.-S. Chang et al.
Biomarkers, 2013; 18(8): 716–720
Biomarkers Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
Table 1. Clinical characteristics and biochemical variables.
Demographic data Age (years) Body mass index (kg/m2) Smoking index (pack*year) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Lipid profile Cholesterol (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) Triglyceride (mg/dL) Cytokines MMP-9 (ng/ml) MMP-2 (ng/ml) CyP-A (ng/ml) sE-seletin (mg/dL) sICAM-1 (mg/dL) sVCAM-1 (mg/dL) IL-2 (pg/mL) IL-4 (pg/mL) NOx (mmol/L)
Hypertension group (n ¼ 40)
Non-hypertension group (n ¼ 40)
p Value
58 4.1 25.1 2.3 8.7 21.7 119 13 75 15
56 10.2 23.0 2.8 2.3 7.6 154 10 96 7
0.140a 0.001a* 0.065a 0.001a 0.001a
207.0 35.0 54.6 61.2 126.0 31.0 132.0 60.3
193.5 36.7 61.2 15.1 113.4 32.9 97.5 57.3
0.036a* 0.005a* 0.028a* 0.001a*
33.3 24 256.2 53.1 94.5 26.2 30.4 30.1 264.8 71.9 514.7 184.9 0.4 0.7 0.5 1.4 133.1 46.2
31.5 23.6 268.3 82.5 73.1 29.8 23.5 21.1 266.7 93.2 461.3 140.5 0.6 2.4 0.6 1.4 136.2 54.4
0.693b 0.468b 0.001b* 0.189b 0.908b 0.077b 0.585b 0.860b 0.766b
a Paired t test, bANOVA under control of BMI, smoking index and lipid profile. *p50.05.
Table 2. Regression model for the analysis of interfering factors in subjects with or without hypertension. Variables in the equation Body mass index Smoking index Cholesterol LDL-C HDL-C Triglyceride CyP-A
B
SE
Wald
p Value
0.278 0.026 0.130 0.133 0.128 0.020 0.022
0.121 0.017 0.287 0.286 0.287 0.057 0.009
5.262 2.297 0.207 0.216 0.198 0.128 5.380
0.022* 0.130 0.649 0.642 0.656 0.720 0.020*
LDL-C, low-density lipoproteins cholesterol; HDL-C, high-density lipoproteins cholesterol. *p50.05.
subjects showed a positive correlation with systolic blood pressure (R2 ¼ 0.019, p ¼ 0.029) and DBP (R2 ¼ 0.017, p ¼ 0.047) under control of BMI, smoking index and lipid profile (Figure 1). No correlations among BMI, lipid profile, and CyP-A were disclosed in either the hypertension or non-hypertension groups.
Discussion The main original finding of the present study was that the plasma CyP-A level was higher in the subjects with hypertension than in the non-hypertension males. To the best of our knowledge, this was the first study to identify plasma Cyp-A as the hypertension-related biomarker in vivo. The validity of our results was strengthened by the agematched case-control study design. Plasma CyP-A was the only cytokine exhibiting a significantly positive correlation with systolic blood pressure and diastolic blood pressure. CyP-A is a 20 kD chaperone protein secreted from vascular smooth muscle cells in response to reactive oxygen species (ROS) (Jin et al., 2000). It contributes to inflammation and
atherosclerosis by promoting endothelial cell apoptosis and expression of leukocyte adhesion molecules in both intracellular and extracellular space (Satoh et al., 2008; Nigro et al., 2011). CyP-A also plays a crucial role in stimulating T helper cells with type 1 responses (Arora et al., 2005), up-regulating MMP activation (Yang et al., 2008), and increasing transduction of pro-inflammatory signals in vascular smooth muscle cells and proliferation of macrophages in vitro and in vivo (Satoh et al., 2008, 2010). CyP-A augments AngiotensinII-induced ROS production and inflammatory cell recruitment into arterial vascular smooth muscle cells (Satoh et al., 2009). Nigro et al. (2011) demonstrated that extracellular CyP-A would stimulate the cell adhesion molecules expression in endothelial cells and enhance extracellular proliferation and angiogenesis. Focal accumulation of lipoproteins, monocyte-derived macrophages, and lymphocytes within the arterial wall are in favor of atherosclerotic formation. These contribute to the progression of atherosclerotic plaques and promote vulnerable lesions (Chrysohoou et al., 2004). Therefore, CyP-A plays an important role in inflammatory cell recruitment/migration and vascular smooth muscle cells proliferation, resulting in hypertensive vascular diseases in vivo (Yang et al., 2008). To identify the risk factors contributing to hypertension, multiple linear regression analysis was performed. We found that smoking index, cholesterol, LDL-C, HDL-C, and triglyceride did not contribute significantly to hypertension. However, BMI and plasma CyP-A did promote hypertension considerably. BMI is positively and independently associated with morbidity and mortality from hypertension, cardiovascular disease, cerebral stroke, type II diabetes mellitus and other chronic diseases (Pi-Sunyer, 1993). Many studies have quantified and performed powerful analysis of the link between hypertension and increased BMI and abnormal lipids, and these studies have indicated that control and management of raised blood pressure and cholesterol are
CyP-A for untreated male essential hypertension
DOI: 10.3109/1354750X.2013.847122
Biomarkers Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
(a)
719
(b)
Figure 1. Plot of plasma CyP-A and systolic blood pressure (a) and diastolic blood pressure (b) under partial correlation analysis with BMI, smoking index and lipid profile control.
specifically important for obese people with hypertension (Brown et al., 2000; Kummayinka, 1997). No differences in circulating biomarkers, including MMP2, MMP-9, sE-selectin, sICAM, sVCAM, IL-2, IL-4 and NOx, between the hypertensive and non-hypertensive subjects were found in the present study. The untreated hypertensive patients enrolled in this study had no other inflammatory risk factors for vascular diseases, except a higher BMI and mild hyperlipidemia, which allowed us to exclude any illnessrelated influences on the assessed parameters. This was a pilot age-matched case-control study in untreated hypertensive patients. Potential limitations of the present study include that the study was not prospective and small scale. In the future, we will recruit a larger sample and perform cross-sectional prospective and animal studies to validate our findings related to the possible role of CyP-A in hypertension. In summary, our findings demonstrated that the plasma level of CyP-A significantly corresponds to alteration of systolic/diastolic blood pressure, and could be a valuable and critical biomarker for the initial assessment of early hypertensive vascular disease.
Acknowledgements This project was supported by Changhua Christian Hospital, Changhua, Taiwan.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by grants 97-CCH-ICO-19-2, 97-CCH-ICO-21-2 and 97-CCH-ICO-20-2 from Changhua Christian Hospital, Changhua, Taiwan.
References Arora K, Gwinn WM, Bower MA, et al. (2005). Extracellular cyclophilins contribute to the regulation of inflammatory responses. J Immunol 175:517–22. Bermudez EA, Rifai N, Buring J, et al. (2002). Interrelationships among circulating interleukin (IL)-6, C-reactive protein, and traditional
cardiovascular risk factors in women. Arterioscler Thromb Vasc Biol 22:1668–73. Blann AD, Tse W, Maxwell SJ, Waite MA. (1994). Increased levels of the soluble adhesion molecule E-selectin in essential hypertension. J Hypertens 12:925–8. Brown CD, Higgins M, Donato KA, et al. (2000). Body mass index and the prevalence of hypertension and dyslipidemia. Obes Res 8: 605–19. Chrysohoou C, Pitsavos C, Panagiotakos DB, et al. (2004). Association between prehypertension status and inflammatory markers related to atherosclerotic disease: the ATTICA study. Am J Hypertens 17: 568–73. Davey Smith G, Lawlor DA, Harbord R, et al. (2005). Association of c-reactive protein with blood pressure and hypertension. Arteriosclerosis Thromb Vascular Biol 25:1051–60. Friedewald WT, Levy RI, Fredrickson DS. (1972). Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 8: 499–502. Green LC, Wagner DA, Glogowski J, et al. (1982). Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 126: 131–8. Handschumacher RE, Harding MW, Rice J, et al. (1984). Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226: 544–7. Hwang SJ, Ballantyne CM, Sharrett AR, et al. (1997). Circulating adhesion molecules VCAM-a, ICAM-1, and E-selectin in carotid atherosclerosis and incident coronary heart disease cases. Circulation 96:4219–25. Iiyama K, Nagano M, Yo Y, et al. (1996). Impaired endothelial function with essential hypertension assessed by ultrasonography. Am Heart J 132:779–82. Jin ZG, Melaragno MG, Liao DF, et al. (2000). Cyclophilin A is a secreted growth factor induced by oxidative stress. Circ Res 87: 789–96. Kato N, Sugiyama T, Morita H, et al. (1999). Lack of evidence for association between the eNOS gene and hypertension. Hypertension 33:933–6. Komatsu S, Pane´s J, Russell JM, et al. (1997). Effects of chronic arterial hypertension on constitutive and induced intercellular adhesion molecule-1 expression in vivo. Hypertension 29:683–9. Kummayinka SK. (1997). The impact of obesity on hypertension management in African Americans. J Healthcare Poor Underserved 8: 352–64. Kuroda YT, Komamura K, Tatsumi R, et al. (2001). Vascular cell adhesion molecule-1 as a biochemical marker of left ventricular mass in the patients with hypertension. Am J Hypertens 14:868–72. Lacy F, O’Connor DT, Schmid-Schonbein GW. (1998). Plasma hydrogen peroxide production in hypertensives and normotensive subjects at enetic risk of hypertension. J Hypertension 16:291–303.
Biomarkers Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
720
C.-S. Chang et al.
Lim CS, Shalhoub J, Gohel MS, et al. (2010). Matrix metalloproteinases in vascular disease–a potential therapeutic target? Curr Vasc Pharmacol 8:75–85. Li-Saw-Hee FL, Edmunds E, Blann AD, et al. (2000). Matrix metalloproteinase-9 and tissue inhibitor metalloproteinase-1 levels in essential hypertension. Relationship to left ventricular mass and anti-hypertensive therapy. Int J Cardiol 75:43–7. McGowan MW, Artiss JD, Strandbergh DR, Zak B. (1983). A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin Chem 29:538–42. Miller MA, Kerry SM, Cook DG, Cappuccio FP. (2004). Cellular adhesion molecules and blood pressure interaction with sex in a multiethnic population. J Hypertens 22:705–11. Nigro P, Satoh K, O’Dell MR, et al. (2011). Cyclophilin A is an inflammatory mediator that promotes atherosclerosis in apolipoprotein E-deficient mice. J Exp Med 208:53–66. Oparil S, Zaman MA, Calhoun DA. (2003). Pathogenesis of hypertension. AnnIntern Med 139:761–76. Olsen MH, Wachtell K, Aalkjaer C, et al. (2002). Vasodilatory capacity and vascular structure in long-standing hypertension: a LIFE substudy. Losartan Intervention For Endpoint-Reduction in Hypertension. Am J Hypertens 15:398–404. Onal IK, Altun B, Onal ED, et al. (2009). Serum levels of MMP-9 and TIMP-1 in primary hypertension and effect of antihypertensive treatment. Eur J Intern Med 20:369–72.
Biomarkers, 2013; 18(8): 716–720
Panek AN, Bader M. (2006). Matrix reloaded: the matrix metalloproteinase paradox. Hypertension 47:640–1. Pi-Sunyer FX. (1993). Medical hazards of obesity. Ann Intern Med 119: 655–60. Satoh K, Matoba T, Suzuki J, et al. (2008). Cyclophilin A mediates vascular remodeling by promoting inflammation and vascular smooth muscle cell proliferation. Circulation 117: 3088–98. Satoh K, Nigro P, Berk BC. (2010). Oxidative stress and vascular smooth muscle cell growth: a mechanistic linkage by cyclophilin A. Antioxid Redox Signal 12:675–82. Satoh K, Nigro P, Matoba T, et al. (2009). Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II-induced aortic aneurysms. Nat Med 15:649–56. Tuttle RS, Boppana DP. (1990). Antihypertensive effect of interleukin-2. Hypertension 15:89–94. Touyz RM. (2000). Oxidative stress and vascular damage in hypertension. Curr Hypertens Rep 2:98–105. Warnick GR, Benderson J, Albers JJ. (1982). Dextran sulfate-Mg2þ precipitation procedure for quantitation of high-density-lipoprotein cholesterol. Clin Chem 28:1379–88. Yang Y, Lu N, Zhou J, et al. (2008). Cyclophilin A up-regulates MMP-9 expression and adhesion of monocytes/macrophages via CD147 signalling pathway in rheumatoid arthritis. Rheumatology (Oxford) 47:1299–310.
RESEARCH ARTICLE
Cyclophilin-A: a novel biomarker for untreated male essential hypertension Chen-Shu Chang1,2, Shih-Li Su3,4, Chia-Chu Chang5, Kwo-Whei Lee6, Chen-Ling Kuo7, Ching-Shan Huang7, Wan-Min Tseng7, and Chin-San Liu1,7,8 Department of Neurology, Changhua Christian Hospital, Changhua, Taiwan, 2Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science of Technology, Taichung, Taiwan, 3Division of Endocrinology and Metabolism, Department of Internal Medicine, Diabetes Education Center, Changhua Christian Hospital, Changhua, Taiwan, 4Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan, 5Division of Nephrology, Department of Internal Medicine, 6Department of Medical Imaging, 7Vascular and Genomic Research Center, Changhua Christian Hospital, Changhua, Taiwan, and 8Graduate Institute of Integrative Medicine, China Medical University, Taichung, Taiwan Abstract
Keywords
Vascular cytokines, total nitrite, and cyclophilin-A (CyP-A) may be related to the pathogenesis of untreated hypertension. Forty males with normotensive and untreated essential hypertension were recruited in this cytokines survey. Body mass index (BMI), hyperlipidemia, and plasma CyP-A were increased in the hypertensive group (p50.05). However, only BMI (p ¼ 0.022) and plasma CyP-A (p ¼ 0.020) were found to be significant contributors to hypertension by multiple regression analysis. CyP-A was also positively correlated with systolic blood pressure (p ¼ 0.029) and diastolic blood pressure (p ¼ 0.047). These findings indicated that plasma CyP-A is a critical molecular biomarker in the early pathogenesis of essential hypertension.
Atherosclerosis, cyclophilin-A, cytokine, hypertension
Introduction The pathogenesis of arterial hypertension is multifactorial (Oparil et al., 2003). Major physiological alterations have been identified in hypertensive individuals, including vascular hypertrophy (Olsen et al., 2002), systemic inflammation (Bermudez et al., 2002), endothelial dysfunction (Iiyama et al., 1996), and enhanced oxidative stress (Lacy et al., 1998). These mechanisms ordinarily occur in an insidious and asymptomatic pattern over the development course of hypertension. Several cytokines have been found to be involved in the pathogenesis of hypertension-related atherosclerosis. Vascular endothelial function can be assessed by matrix metalloproteinase (MMP) in many cardiovascular diseases including hypertension and its chronic complications (Lim et al., 2010). Increased vascular expressions of MMP-2 and MMP-9 were found in hypertensive subjects (Li-Saw-Hee et al., 2000; Onal et al., 2009), but another study reported decreased MMP-2 and MMP-9 in hypertensive individuals (Panek & Bader, 2006). Biomarkers of endothelial cell activation are generally surveyed in hypertension, including adhesion molecule soluble endothelium-selectin (sE-selectin) (Blann et al., 1994), soluble intercellular adhesion molecule-1 Address for correspondence: Chin-San Liu, MD, PhD, Department of Neurology, Vascular and Genomic Research Center, Changhua Christian Hospital, 135 Nanhsiao Street, Changhua 500, Taiwan. Tel: +886-47238595 ext.4752. Fax: +886-4-7238595 ext.4063. E-mail: liu48111@ gmail.com
History Received 13 August 2013 Revised 13 September 2013 Accepted 17 September 2013 Published online 28 October 2013
(sICAM-1) (Komatsu et al., 1997), and soluble vascular cell adhesion molecule-1 (sVCAM-1) (Kuroda et al., 2001), but there are inconsistencies in the published literature (Hwang et al., 1997; Miller et al., 2004). Elevation of the concentrations of plasma interleukin-2 (IL-2), interleukin-4 (IL-4) (Tuttle & Boppana, 1990) and total nitrite/nitrate concentrations (NOx) (Touyz, 2000) was found in several basic and clinical studies of hypertension, but there some disputed and controversial results (Davey Smith et al., 2005; Kato et al., 1999). Cyclophilin-A (CyP-A), the most abundant form of cyclophilin in human intracellular protein, is secreted by monocytes/macrophages, endothelial cells, and vascular smooth muscle cells in inflammatory and oxidative stimuli in arterial atherosclerosis (Jin et al., 2000; Satoh et al., 2008). CyP-A can elicit leukocyte chemotaxis with raised levels in inflammatory tissues in vitro, was demonstrated to promote atherosclerosis by increasing low-density lipoprotein (LDL) uptake and stimulating recruitment of inflammatory cells in the vessel wall with an oxidative stress environment of the Apoe/Ppia þ/þ mice (Nigro et al., 2011). In the present study, CyP-A was also recruited for potential cytokine screening as a biomarker of hypertension due to its characteristic of inflammatory responses in vascular system (Arora et al., 2005; Handschumacher et al., 1984; Jin et al., 2000; Satoh et al., 2008). The above-mentioned cytokines are all possibly involved in the pathogenesis of essential hypertension during the initial stage. Thus, cytokine biomarkers in response to inflammation-related atherosclerosis are of
20 13
Biomarkers Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
1
DOI: 10.3109/1354750X.2013.847122
interest in the evaluation of the early stage of essential hypertension before a symptomatic event. Therefore, our hypothesis is that alterations of the plasma levels of MMP-2, MMP-9, sE-selectin, sICAM, sVCAM, IL-2, IL-4, NOx, and CyP-A may occur in the early pathogenesis of essential hypertension.
CyP-A for untreated male essential hypertension
717
dihydrochloride) as described by Green et al. (1982). The assay requires conversion of nitrate to nitrite using nitrate reductase, then Griess reagent is added, which converts nitrite into a deep-purple azo compound. Absorption measurements were carried out at 540 nm using a microplate reader. Biochemical measurements
Methods
Biomarkers Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
Study subjects Forty healthy non-hypertensive males and 40 age-matched untreated males with essential hypertension were recruited from the health clinic of Changhua Christian Hospital from September 2009 to October, 2010. The untreated essential hypertension group was defined as those without medication with an average home systolic blood pressure greater than 140 mmHg and a diastolic blood pressure greater than 90 mmHg according to the definition of stage I/II hypertension in the JNC-7 guidelines. All subjects were in good health and free of any cardiovascular diseases as assessed by their medical history, physical examination and routine screening laboratory data taken by an internist and family doctors. Subjects without alcoholism and diabetes (fasting blood glucose less than 110 mg/dL), except for some with mild hyperlipidemia, were recruited for the study. Subjects actively taking medication were excluded. The smoking history of the study subjects was obtained by questionnaire. Passive smoking exposure was not considered in this study, and our nonsmokers had no previous smoking experience. All of the human experimental procedures followed the ethical standards of Changhua Christian Hospital and were approved by the institutional review committee. Informed consent for study enrollment was obtained from each of the subjects. Blood samples were taken from each of the subjects in the morning after he or she had fasted overnight and before smoking if he or she was a smoker. Ten milliliters of whole blood were withdrawn from an antecubital vein. Plasma and serum were immediately collected by centrifugation, divided into several aliquots, and stored at 80 C until analysis. Plasma cytokines measurement The quality of samples in 80 C refrigerator was stable after storage of recruited whole blood within 5 months. Three samples were repeatedly measured in the estimation of all cytokines and lipid profiles at 1st day and 3rd month of samples collection. No statistic difference was observed in this pilot calibration study in the same sample after repeated measurement with 3 months. All biochemical studies of samples would be performed with 3 months. Plasma CyP-A was measured using commercially-available enzyme-linked immunosorbent assay kits (Wuhan USCN Science, Wuhan, China) in accordance with the manufacturer’s instructions. Plasma total MMP-2 and MMP-9 levels were measured by ELISA (R&D Systems, Minneapolis, MN). Plasma IL-2 and IL-4 levels were measured by ELISA (eBioscience, San Diego, CA) according to the manufacturer’s instructions. Plasma sVCAM-1 and sE-selectin levels were measured by ELISA (R&D Systems). The plasma NOx level was measured by Griess reagent (0.1% N-(1-naphthyl) ethylenediamine
Total serum cholesterol was determined enzymatically using the Sigma Diagnostics cholesterol reagent (Sigma Chemical Co., St. Louis, MO). Serum high density lipoprotein cholesterol (HDL-C) was determined using the Sigma Diagnostics HDL-C reagent (Warnick et al., 1982). Dextran sulfate and magnesium ions were used for precipitation and isolation of LDL and very LDL particles. Serum triglyceride concentrations were determined using the Sigma Diagnostics triglyceride reagent following the modification described by McGowan et al. (1983). LDL-C concentration was calculated according to the formula developed by Friedewald et al. (1972). Statistical analysis Data are expressed as the mean SD. All statistical analyses were performed using the SPSS statistical package, version 17.0 (Chicago, IL). The significances of the differences between the mean values in the non-hypertension and hypertension groups were determined by paired t-test and one-way analysis of variance (ANOVA) under control of BMI, smoking index, and lipid profile. Multiple linear regression was applied to assess the importance of various contributing factors to hypertension. Partial correlation was applied in the comparison between blood pressure and the plasma level of CyP-A under control of BMI, smoking index, and lipid profile.
Results The baseline demographic data of the 40 hypertensive and 40 non-hypertensive gentlemen enrolled in this study are shown in Table 1. The hypertension group revealed a higher body mass index (BMI) than the non-hypertension group (25.1 2.3 versus 23.0 2.8; p ¼ 0.001). The hypertension group had higher cholesterol (207.0 35.0 versus 193.5 36.7, p ¼ 0.036), LDL-C (126.0 31.0 versus 113.4 32.9, p ¼ 0.028) and triglyceride (132.0 60.3 versus 97.5 57.3, p ¼ 0.001) than the non-hypertension group. In addition, HDL-C (54.6 61.2 versus 61.2 15.1, p ¼ 0.005) was decreased in the non-hypertension group. Additionally, plasma CyP-A was higher in the hypertension group than in the non-hypertension group (94.5 26.2 versus 73.1 29.8; p ¼ 0.001). However, blood pressure had no effect on the other biochemical data, including MMP-2, MMP-9, adhesion molecules, IL-2, IL-4, and nitric oxide (NOx). To assess the significant factors contributing to hypertension shown in Table 1, multiple linear regression analysis was applied, and the results are presented in Table 2. We found that smoking index, cholesterol, LDL-C, HDL-C, and triglyceride level did not contribute significantly to hypertension, except BMI (p ¼ 0.022) and plasma CyP-A (p ¼ 0.020). The increased CyP-A concentration in the study
718
C.-S. Chang et al.
Biomarkers, 2013; 18(8): 716–720
Biomarkers Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
Table 1. Clinical characteristics and biochemical variables.
Demographic data Age (years) Body mass index (kg/m2) Smoking index (pack*year) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Lipid profile Cholesterol (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) Triglyceride (mg/dL) Cytokines MMP-9 (ng/ml) MMP-2 (ng/ml) CyP-A (ng/ml) sE-seletin (mg/dL) sICAM-1 (mg/dL) sVCAM-1 (mg/dL) IL-2 (pg/mL) IL-4 (pg/mL) NOx (mmol/L)
Hypertension group (n ¼ 40)
Non-hypertension group (n ¼ 40)
p Value
58 4.1 25.1 2.3 8.7 21.7 119 13 75 15
56 10.2 23.0 2.8 2.3 7.6 154 10 96 7
0.140a 0.001a* 0.065a 0.001a 0.001a
207.0 35.0 54.6 61.2 126.0 31.0 132.0 60.3
193.5 36.7 61.2 15.1 113.4 32.9 97.5 57.3
0.036a* 0.005a* 0.028a* 0.001a*
33.3 24 256.2 53.1 94.5 26.2 30.4 30.1 264.8 71.9 514.7 184.9 0.4 0.7 0.5 1.4 133.1 46.2
31.5 23.6 268.3 82.5 73.1 29.8 23.5 21.1 266.7 93.2 461.3 140.5 0.6 2.4 0.6 1.4 136.2 54.4
0.693b 0.468b 0.001b* 0.189b 0.908b 0.077b 0.585b 0.860b 0.766b
a Paired t test, bANOVA under control of BMI, smoking index and lipid profile. *p50.05.
Table 2. Regression model for the analysis of interfering factors in subjects with or without hypertension. Variables in the equation Body mass index Smoking index Cholesterol LDL-C HDL-C Triglyceride CyP-A
B
SE
Wald
p Value
0.278 0.026 0.130 0.133 0.128 0.020 0.022
0.121 0.017 0.287 0.286 0.287 0.057 0.009
5.262 2.297 0.207 0.216 0.198 0.128 5.380
0.022* 0.130 0.649 0.642 0.656 0.720 0.020*
LDL-C, low-density lipoproteins cholesterol; HDL-C, high-density lipoproteins cholesterol. *p50.05.
subjects showed a positive correlation with systolic blood pressure (R2 ¼ 0.019, p ¼ 0.029) and DBP (R2 ¼ 0.017, p ¼ 0.047) under control of BMI, smoking index and lipid profile (Figure 1). No correlations among BMI, lipid profile, and CyP-A were disclosed in either the hypertension or non-hypertension groups.
Discussion The main original finding of the present study was that the plasma CyP-A level was higher in the subjects with hypertension than in the non-hypertension males. To the best of our knowledge, this was the first study to identify plasma Cyp-A as the hypertension-related biomarker in vivo. The validity of our results was strengthened by the agematched case-control study design. Plasma CyP-A was the only cytokine exhibiting a significantly positive correlation with systolic blood pressure and diastolic blood pressure. CyP-A is a 20 kD chaperone protein secreted from vascular smooth muscle cells in response to reactive oxygen species (ROS) (Jin et al., 2000). It contributes to inflammation and
atherosclerosis by promoting endothelial cell apoptosis and expression of leukocyte adhesion molecules in both intracellular and extracellular space (Satoh et al., 2008; Nigro et al., 2011). CyP-A also plays a crucial role in stimulating T helper cells with type 1 responses (Arora et al., 2005), up-regulating MMP activation (Yang et al., 2008), and increasing transduction of pro-inflammatory signals in vascular smooth muscle cells and proliferation of macrophages in vitro and in vivo (Satoh et al., 2008, 2010). CyP-A augments AngiotensinII-induced ROS production and inflammatory cell recruitment into arterial vascular smooth muscle cells (Satoh et al., 2009). Nigro et al. (2011) demonstrated that extracellular CyP-A would stimulate the cell adhesion molecules expression in endothelial cells and enhance extracellular proliferation and angiogenesis. Focal accumulation of lipoproteins, monocyte-derived macrophages, and lymphocytes within the arterial wall are in favor of atherosclerotic formation. These contribute to the progression of atherosclerotic plaques and promote vulnerable lesions (Chrysohoou et al., 2004). Therefore, CyP-A plays an important role in inflammatory cell recruitment/migration and vascular smooth muscle cells proliferation, resulting in hypertensive vascular diseases in vivo (Yang et al., 2008). To identify the risk factors contributing to hypertension, multiple linear regression analysis was performed. We found that smoking index, cholesterol, LDL-C, HDL-C, and triglyceride did not contribute significantly to hypertension. However, BMI and plasma CyP-A did promote hypertension considerably. BMI is positively and independently associated with morbidity and mortality from hypertension, cardiovascular disease, cerebral stroke, type II diabetes mellitus and other chronic diseases (Pi-Sunyer, 1993). Many studies have quantified and performed powerful analysis of the link between hypertension and increased BMI and abnormal lipids, and these studies have indicated that control and management of raised blood pressure and cholesterol are
CyP-A for untreated male essential hypertension
DOI: 10.3109/1354750X.2013.847122
Biomarkers Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
(a)
719
(b)
Figure 1. Plot of plasma CyP-A and systolic blood pressure (a) and diastolic blood pressure (b) under partial correlation analysis with BMI, smoking index and lipid profile control.
specifically important for obese people with hypertension (Brown et al., 2000; Kummayinka, 1997). No differences in circulating biomarkers, including MMP2, MMP-9, sE-selectin, sICAM, sVCAM, IL-2, IL-4 and NOx, between the hypertensive and non-hypertensive subjects were found in the present study. The untreated hypertensive patients enrolled in this study had no other inflammatory risk factors for vascular diseases, except a higher BMI and mild hyperlipidemia, which allowed us to exclude any illnessrelated influences on the assessed parameters. This was a pilot age-matched case-control study in untreated hypertensive patients. Potential limitations of the present study include that the study was not prospective and small scale. In the future, we will recruit a larger sample and perform cross-sectional prospective and animal studies to validate our findings related to the possible role of CyP-A in hypertension. In summary, our findings demonstrated that the plasma level of CyP-A significantly corresponds to alteration of systolic/diastolic blood pressure, and could be a valuable and critical biomarker for the initial assessment of early hypertensive vascular disease.
Acknowledgements This project was supported by Changhua Christian Hospital, Changhua, Taiwan.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This study was supported by grants 97-CCH-ICO-19-2, 97-CCH-ICO-21-2 and 97-CCH-ICO-20-2 from Changhua Christian Hospital, Changhua, Taiwan.
References Arora K, Gwinn WM, Bower MA, et al. (2005). Extracellular cyclophilins contribute to the regulation of inflammatory responses. J Immunol 175:517–22. Bermudez EA, Rifai N, Buring J, et al. (2002). Interrelationships among circulating interleukin (IL)-6, C-reactive protein, and traditional
cardiovascular risk factors in women. Arterioscler Thromb Vasc Biol 22:1668–73. Blann AD, Tse W, Maxwell SJ, Waite MA. (1994). Increased levels of the soluble adhesion molecule E-selectin in essential hypertension. J Hypertens 12:925–8. Brown CD, Higgins M, Donato KA, et al. (2000). Body mass index and the prevalence of hypertension and dyslipidemia. Obes Res 8: 605–19. Chrysohoou C, Pitsavos C, Panagiotakos DB, et al. (2004). Association between prehypertension status and inflammatory markers related to atherosclerotic disease: the ATTICA study. Am J Hypertens 17: 568–73. Davey Smith G, Lawlor DA, Harbord R, et al. (2005). Association of c-reactive protein with blood pressure and hypertension. Arteriosclerosis Thromb Vascular Biol 25:1051–60. Friedewald WT, Levy RI, Fredrickson DS. (1972). Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 8: 499–502. Green LC, Wagner DA, Glogowski J, et al. (1982). Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem 126: 131–8. Handschumacher RE, Harding MW, Rice J, et al. (1984). Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226: 544–7. Hwang SJ, Ballantyne CM, Sharrett AR, et al. (1997). Circulating adhesion molecules VCAM-a, ICAM-1, and E-selectin in carotid atherosclerosis and incident coronary heart disease cases. Circulation 96:4219–25. Iiyama K, Nagano M, Yo Y, et al. (1996). Impaired endothelial function with essential hypertension assessed by ultrasonography. Am Heart J 132:779–82. Jin ZG, Melaragno MG, Liao DF, et al. (2000). Cyclophilin A is a secreted growth factor induced by oxidative stress. Circ Res 87: 789–96. Kato N, Sugiyama T, Morita H, et al. (1999). Lack of evidence for association between the eNOS gene and hypertension. Hypertension 33:933–6. Komatsu S, Pane´s J, Russell JM, et al. (1997). Effects of chronic arterial hypertension on constitutive and induced intercellular adhesion molecule-1 expression in vivo. Hypertension 29:683–9. Kummayinka SK. (1997). The impact of obesity on hypertension management in African Americans. J Healthcare Poor Underserved 8: 352–64. Kuroda YT, Komamura K, Tatsumi R, et al. (2001). Vascular cell adhesion molecule-1 as a biochemical marker of left ventricular mass in the patients with hypertension. Am J Hypertens 14:868–72. Lacy F, O’Connor DT, Schmid-Schonbein GW. (1998). Plasma hydrogen peroxide production in hypertensives and normotensive subjects at enetic risk of hypertension. J Hypertension 16:291–303.
Biomarkers Downloaded from informahealthcare.com by University of Toronto on 08/11/14 For personal use only.
720
C.-S. Chang et al.
Lim CS, Shalhoub J, Gohel MS, et al. (2010). Matrix metalloproteinases in vascular disease–a potential therapeutic target? Curr Vasc Pharmacol 8:75–85. Li-Saw-Hee FL, Edmunds E, Blann AD, et al. (2000). Matrix metalloproteinase-9 and tissue inhibitor metalloproteinase-1 levels in essential hypertension. Relationship to left ventricular mass and anti-hypertensive therapy. Int J Cardiol 75:43–7. McGowan MW, Artiss JD, Strandbergh DR, Zak B. (1983). A peroxidase-coupled method for the colorimetric determination of serum triglycerides. Clin Chem 29:538–42. Miller MA, Kerry SM, Cook DG, Cappuccio FP. (2004). Cellular adhesion molecules and blood pressure interaction with sex in a multiethnic population. J Hypertens 22:705–11. Nigro P, Satoh K, O’Dell MR, et al. (2011). Cyclophilin A is an inflammatory mediator that promotes atherosclerosis in apolipoprotein E-deficient mice. J Exp Med 208:53–66. Oparil S, Zaman MA, Calhoun DA. (2003). Pathogenesis of hypertension. AnnIntern Med 139:761–76. Olsen MH, Wachtell K, Aalkjaer C, et al. (2002). Vasodilatory capacity and vascular structure in long-standing hypertension: a LIFE substudy. Losartan Intervention For Endpoint-Reduction in Hypertension. Am J Hypertens 15:398–404. Onal IK, Altun B, Onal ED, et al. (2009). Serum levels of MMP-9 and TIMP-1 in primary hypertension and effect of antihypertensive treatment. Eur J Intern Med 20:369–72.
Biomarkers, 2013; 18(8): 716–720
Panek AN, Bader M. (2006). Matrix reloaded: the matrix metalloproteinase paradox. Hypertension 47:640–1. Pi-Sunyer FX. (1993). Medical hazards of obesity. Ann Intern Med 119: 655–60. Satoh K, Matoba T, Suzuki J, et al. (2008). Cyclophilin A mediates vascular remodeling by promoting inflammation and vascular smooth muscle cell proliferation. Circulation 117: 3088–98. Satoh K, Nigro P, Berk BC. (2010). Oxidative stress and vascular smooth muscle cell growth: a mechanistic linkage by cyclophilin A. Antioxid Redox Signal 12:675–82. Satoh K, Nigro P, Matoba T, et al. (2009). Cyclophilin A enhances vascular oxidative stress and the development of angiotensin II-induced aortic aneurysms. Nat Med 15:649–56. Tuttle RS, Boppana DP. (1990). Antihypertensive effect of interleukin-2. Hypertension 15:89–94. Touyz RM. (2000). Oxidative stress and vascular damage in hypertension. Curr Hypertens Rep 2:98–105. Warnick GR, Benderson J, Albers JJ. (1982). Dextran sulfate-Mg2þ precipitation procedure for quantitation of high-density-lipoprotein cholesterol. Clin Chem 28:1379–88. Yang Y, Lu N, Zhou J, et al. (2008). Cyclophilin A up-regulates MMP-9 expression and adhesion of monocytes/macrophages via CD147 signalling pathway in rheumatoid arthritis. Rheumatology (Oxford) 47:1299–310.
