doi: 10.1161/hy0302.104670
(Hypertension. 2002;39:777.)
© 2002 American Heart Association, Inc.
Scientific Contributions |
Oxidative Stress in Leukocytes Is a Possible Link Between Blood Pressure, Blood Glucose, and C-Reacting Protein
From the Department of Cardiology, Graduate School of Medicine, Osaka City University, Japan.
Correspondence to Kenichi Yasunari, MD, PhD, Department of Cardiology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. E-mail yasunari{at}osaka.med.or.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Because oxidative stress and inflammation are believed to play roles in the pathogenesis of cardiovascular diseases, oxidative stress in polymorphonuclear leukocytes (PMNs) and mononuclear cells (MNCs) has been measured. A total of 529 subjects participated this study. Intracellular oxidative stress in PMNs and MNCs was measured by gated flow cytometry using carboxyfluorescin diacetate bis-acetoxymethyl ester. C-reacting protein (CRP), insulin action (homeostasis model assessment), and traditional risk factors such as age, gender, body mass index, triglycerides, LDL cholesterol, HDL cholesterol, hemoglobin A1c, and mean blood pressure were also measured. Multiple regression analysis revealed a significant correlation between mean blood pressure and PMN oxidative stress (r=0.104, P=0.018). It also demonstrated a significant correlation between hemoglobin A1c and PMN oxidative stress (r=0.112, P=0.021). A significant correlation was also found between CRP and MNC oxidative stress (r=0.116, P=0.008) by multiple regression analysis. In patients with both hypertension and diabetes, both PMN and MNC oxidative stress was increased (n=21, P=0.022 and P=0.006). These results suggest that both hypertension and diabetes lead to increased oxidative stress of PMNs and MNCs, and that CRP is related to MNC oxidative stress.
Key Words: leukocytes • arteries • diabetes mellitus • oxidative stress
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Although hypertension and diabetes are well-recognized risk factors for cardiovascular disease,1,2 the links between hypertension or diabetes and oxidative stress are not fully understood. It has recently been shown that patients with hypertension or diabetes tend to have a higher body mass index and tend to be glucose-intolerant, hyperinsulinemic (insulin-resistant), and dyslipidemic, with high plasma triglyceride and low HDL cholesterol concentrations, which are recognized risk factors for cardiovascular disease.3,4 Though these changes may account for the increased cardiovascular risk in patients with hypertension and those with diabetes, the mechanistic links between these syndrome and enhanced oxidative stress in vivo is not known.
Polymorphonuclear leukocytes (PMNs) are one of the main types of inflammatory cells. Once activated, PMNs release reactive oxygen species, including hydrogen peroxide, contributing to endothelial damage and cardiovascular disease.5,6 Monocytes (MNCs) are crucial cells in the genesis of atherosclerotic lesions. MNCs, when stimulated, have been shown to induce adhesion to endothelium, which results in cardiovascular disease.7
In the present study, we have compared PMN and MNC oxidative stress in hypertensive and/or diabetic subjects and evaluated the relationship of each risk factor for cardiovascular diseases and PMN or MNC oxidative stress.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
The study was performed a prospective study of 529 apparently healthy subjects who visited Osaka City University Hospital. The hypertensive group consisted of subjects who had not been treated or those who had stopped antihypertensive agents for 4 weeks with blood pressure
140/90 mm Hg, and the diabetes group consisted of untreated subjects with hemoglobin A 1C (HbA1C) 6.5 mg/dL. Except for hypertension and diabetes, individuals enrolled in the study were determined to be healthy on the basis of medical history, physical examination, and results of routine laboratory tests. The study protocol was approved by the Osaka City University Institutional Review Board, and written informed consent was obtained from all subjects. All studies were performed after an overnight fast. Degree of obesity was estimated by body mass index. Venous blood was used for measurement of plasma insulin, plasma glucose, plasma C-reacting protein (CRP), plasma cholesterol, triglyceride, and HDL cholesterol concentrations. Serum CRP was measured by a highly sensitive microparticle enzyme immunoassay. An estimate of insulin resistance by homeostasis model assessment (HOMA-IR) was calculated with the following formula: [fasting serum insulin (µU/mL)x fasting plasma glucose (mmol/L)/22.5].
Hydrogen peroxide production by PMNs and MNCs was measured using a gated flow cytometry derived from the technique described by Bass et al8 with some modifications.9 Fresh blood (1 mL) from participants was preincubated for 15 minutes with 2', 7'-carboxydichlorofluorescein diacetate bis-acetoxymethyl ester (CDCFH bis-AM ester; 100 µmol/l). CDCFH diacetate bis-AM ester is a nonpolar compound that is converted into a nonfluorescent polar derivative (CDCFH) by cellular esterases after incorporation into cells. CDCFH is membrane-impermeable and rapidly oxidized to the highly fluorescent carboxydichlorofluorescein (CDCF) in the presence of intracellular hydrogen peroxide and peroxidases. The oxidative stress in PMNs and MNCs was measured as fluorescence intensity by gated flow cytometry.
Valves are expressed as mean±SD, unless otherwise specified. Student’s unpaired t test was used to compare groups, preceded by 1-way ANOVA. The relationship between PMN or MNC oxidative stress and relevant covariates was examined by determination of standardized correlation coefficients and multiple regression analysis.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Blood Pressure
All subjects were divided into hypertensive group (HT; BP
140/90 mm Hg, n=219) and normotensive group (NT; BP <140/90 mm Hg, n=310) Baseline characteristics of the 2 experimental groups are shown in Table 1. PMN oxidative stress was significantly increased in cell from the HT group. None of the other differences between measured variables in the 2 groups were significant. The relationship between MBP and oxidative stress in PMNs or MNCs is illustrated in Figure 1 and indicates that the higher the blood pressure, the greater the PMN oxidative stress.
|
|
Blood Glucose (HbA1C)
All subjects were divided into a diabetes group (DM; HbA1C 6.5%, n=41) or a nondiabetic group (Non-DM; HbA1C <6.5%, n=488). Baseline characteristics of the 2 experimental groups are shown in Table 2. MNC oxidative stress was significantly increased in the diabetes group. Significant difference was observed between the 2 groups in MNC oxidative stress, HDL cholesterol, HOMA-IR, and insulin (Table 2). The relationship between HbA1C and PMN or MNC oxidative stress is shown in Figure 2 and indicates that the higher HbA1C, the greater the PMN oxidative stress. This relationship was also observed between HbA1C and MNC oxidative stress.
|
|
Hypertensive Diabetic Patients
All subjects were divided into 4 groups: NT and Non-DM (n=290), HT and Non-DM (n=198), HT and DM (n=20), and HT and Non-DM (n=21). Baseline characteristics of the 4 experimental groups are shown in Table 3. Only in HT and DM group, were both PMN and MNC oxidative stress significantly increased; there was also an increase in triglycerides and HOMA-IR, an indicator of insulin resistance, and a decrease in HDL cholesterol.
|
Multiple Regression Analysis
Multiple regression analysis was used to quantify the impact of measured variables on PMN oxidative stress and MNC oxidative stress. The results shown in Table 4 indicate that MBP was significantly related to PMN oxidative stress, and that HbA1C was significantly related to PMN oxidative stress. The results shown in Table 5 indicate that CRP is significantly related to MNC oxidative stress. The relationships between CRP and PMN or MNC oxidative stress are shown in Figure 3. Multiple regression analysis was also used to quantify the impact of measured variables on HOMA-IR, an indicator of insulin resistance. Body mass index (r=0.338, P<0.001, n=529), triglycerides (r=0.149, P=0.004, n=529), and HbA1C (r=0.313, P<0.001, n=529) were significantly related to HOMA-IR. However, neither PMN oxidative stress (P=0.673) nor MNC oxidative stress (P=0.563) was related to HOMA-IR. Oxidative stress in PMNs and MNCs was related (r=0.153, P<0.001, n=529).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
In the present study, we have demonstrated for the first time that only in hypertensive diabetic patients, both PMN and MNC oxidative stress were significantly increased (Table 3). Both hypertension and diabetes are concurrent,10 and the relationship with increased cardiovascular events is suggested.11 Our findings may explain the mechanisms of increased cardiovascular events through the increase in PMN and MNC oxidative stress of hypertensive diabetic subjects.
We have shown that MBP or HbA1C is related to PMN oxidative stress (Table 4), which is consistent with the previous observation.12,13 PMNs contain the identical oxidative stress–generating system to endothelial cells. If they are activated, they would be an additional systemic source of oxidative stress.14
In the present study, we have also demonstrated for the first time that CRP is related to MNC oxidative stress (Tables 5). CRP has been extensively studied as a potential tool for the prediction of cardiovascular events.15 Thus, MNC oxidative stress may play a role as a potential tool for prediction of cardiovascular risk.
Risk factors for cardiovascular diseases such as hypertension, diabetes, and hyperlipidemia are related to both oxidative stress9,16,17 and insulin resistance.18,19 In the present study, neither PMN oxidative stress nor MNC oxidative stress was directly related to HOMA-IR, an indicator of insulin resistance. Multiple regression analysis showed that body mass index, triglycerides, and HbA1C were related to HOMA-IR, suggesting that obesity-related metabolic abnormality was related to insulin resistance.
Possible mechanism of increase in oxidative stress in PMNs and MNCs in relation to hypertension, diabetes, and CRP remains to be elucidated. Pressure and high glucose are reported to activate protein kinase C activation in vitro,9,18 and oxidative stress in PMNs is mediated by protein kinase C.20 Protein kinase C may play a role in the increase in PMN oxidative stress induced by hypertension and diabetes.
In conclusion, we found a significant relationship between PMN oxidative stress and MBP or HbA1C, in addition to a significant relationship between MNC oxidative stress and CRP. We have identified a possible cellular mechanism to explain why oxidative stress in hypertensive and diabetic individuals are increased.
|
Acknowledgments |
|---|
We would like to thank Junko Ohashi and Sayuri Takagi for excellent secretarial assistance. This study was supported by the Ministry of Education, Sports, Science and Technology; Kimura Memorial Heart Foundation; and Research Foundation of Community Medicine.
Received November 14, 2001; first decision December 3, 2001; accepted December 18, 2001.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Kannel WB, Schwartz MJ, McNamara PM. Blood pressure and risk of coronary heart disease. Dis Chest. 1969; 56: 43–52.[Medline] [Order article via Infotrieve]
2.
Stearns S, Schlesinger MJ, Rudy A. Incidence and clinical significance of coronary artery disease in diabetes mellitus. Arch Intern Med. 1947; 80: 463–474.
3.
Fuh MM, Shieh SM, Wu DA, Chen YD, Reaven GM. Abnormalities of carbohydrate and lipid metabolism in patients with hypertension. Arch Intern Med. 1987; 147: 1035–1038.
4.
Shen D-C, Shieh S-M, Fuh M-T, Wu D-A, Chen Y-DI, Reaven GM. Resistance to insulin-stimulated glucose uptake in patients with hypertension. J Clin Endocrinol Metab. 1988; 66: 580–583.
5. Smedly LA, Tonnesen MG, Sandhaus RA, HaslettC , Guthrie LA, Johnston RB Jr, Henson PM, Worthen GS. Neutrophil-mediated injury to endothelial cells: enhancement by endotoxin and essential role of neutrophil elastase. J Clin Invest. 1986; 77: 1233–1243.[Medline] [Order article via Infotrieve]
6. Weiss SJ. Tissue destruction by neutrophils. N Engl J Med. 1989; 320: 365–376.[Medline] [Order article via Infotrieve]
7.
Jialal I, Devaraj S, Kaul N. The effect of 
-tocopherol on monocyte proatherogenic activity. J Nutr. 2001; 131: 389S–394S.
8. Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M. Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol. 1983; 130: 1910–1917.[Abstract]
9.
Yasunari K, Kohno M, Kano H, Minami M, Yokokawa K, Yoshikawa J. Antioxidants improve impaired insulin-mediated glucose uptake and prevent migration and proliferation of cultured rabbit coronary smooth muscle cells induced by high glucose. Circulation. 1999; 99: 1370–1378.
10. The Hypertension in Diabetes Study Group. Hypertension in Diabetes Study (HDS), I: prevalence of hypertension in newly presenting type 2 diabetic patients and the association with risk factors for cardiovascular and diabetic complications. J Hypertens. 1993; 11: 309–317.[CrossRef][Medline] [Order article via Infotrieve]
11.
Grossman E, Messerli FH, Goldbourt U. High blood pressure and diabetes mellitus: are all antihypertensive drugs created equal? Arch Intern Med. 2000; 160: 2447–2452.
12. Kristal B, Shurtz-Swirski R, Chezar J, Manaster J, Levy R, Shaptro G, Weissman I, Shasha SM, Sela S. Involvement of peripheral polymorphonuclear leukocytes in oxidative stress and inflammation in patients with essential hypertension. Am J Hypertens. 1998; 11: 921–928.[CrossRef][Medline] [Order article via Infotrieve]
13.
Shurtz-Swirski R, Sela S, Herskovits AT, Shasha SM, Shapiro G, Nasser L, Kristal B. Involvement of peripheral polymorphonuclear leukocytes in oxidative stress and inflammation in type 2 diabetic patients. Diabetes Care. 2001; 24: 104–110.
14. Pala MG, Paolini G, Paroni R, De Veechi E, Gallorini C, Stefano PL, Di Credico G, Zuccari M, Galli L, Agape V. Myocardial protection with and without leukocyte depletion: a comparative study on the oxidative stress. Eur J Cardiothorac Surg. 1995; 9: 701–706.[Abstract]
15.
Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med. 1997; 336: 973–979.
16. Yasunari K, Maeda K, Nakamura M, Yoshikawa J. Pressure promotes angiotensin II–mediated migration of human coronary smooth muscle cells through increase in oxidative stress. Hypertension, 2002. In press.
17.
Yasunari K, Maeda K, Minami M, Yoshikawa J. HMG-CoA reductase inhibitors prevent migration of human coronary smooth muscle cells through suppression of increase in oxidative stress. Arterioscler Thromb Vasc Biol. 2001; 21: 937–942.
18.
Yasunari K, Kohno M, Kano H, Yokokawa K, Minami M, Yoshikawa J. Mechanisms of action of troglitazone in the prevention of high glucose-induced migration and proliferation of cultured coronary smooth muscle cells. Circ Res. 1997; 81: 953–962.
19. Abe H, Yamada N, Kamata K, Kuwaki T, Shimada M, Osuga J, Shionoiri F, Yahagi N, Kadowaki T, Tamemoto H, Ishibashi S, Yazaki Y, Makuuchi M. Hypertension, hypertriglyceridemia, and impaired endothelium-dependent vascular relaxation in mice lacking insulin receptor substrate-1. J Clin Invest. 1998; 101: 1784–1788.[Medline] [Order article via Infotrieve]
20. Young LH, Ikeda Y, Lefer AM. Protein kinase inhibition exerts cardioprotective effects in myocardial ischemia/reperfusion via inhibition of superoxide release. Methods Find Exp Clin Pharmacol. 2001; 23: 107–114.[CrossRef][Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  L. Guasti, F. Marino, M. Cosentino, R. C. Maio, E. Rasini, M. Ferrari, L. Castiglioni, C. Klersy, G. Gaudio, A. M. Grandi, et al. Prolonged statin-associated reduction in neutrophil reactive oxygen species and angiotensin II type 1 receptor expression: 1-year follow-up Eur. Heart J., May 1, 2008; 29(9): 1118 - 1126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Paravicini and R. M. Touyz NADPH Oxidases, Reactive Oxygen Species, and Hypertension: Clinical implications and therapeutic possibilities Diabetes Care, February 1, 2008; 31(Supplement_2): S170 - S180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Minoguchi, T. Yokoe, A. Tanaka, S. Ohta, T. Hirano, G. Yoshino, C. P. O'Donnell, and M. Adachi Association between lipid peroxidation and inflammation in obstructive sleep apnoea. Eur. Respir. J., August 1, 2006; 28(2): 378 - 385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Agarwal, K. C. Nandipati, R. K. Sharma, C. D. Zippe, and R. Raina Role of Oxidative Stress in the Pathophysiological Mechanism of Erectile Dysfunction J Androl, May 1, 2006; 27(3): 335 - 347. [Full Text] [PDF]
|
|
|
|
 |
|
 D. E. King, A. G. Mainous III, M. E. Geesey, and R. F. Woolson Dietary Magnesium and C-reactive Protein Levels J. Am. Coll. Nutr., June 1, 2005; 24(3): 166 - 171. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Sugano, H. Matsuoka, N. Haramaki, H. Umei, E. Murase, K. Fukami, S. Iida, H. Ikeda, and T. Imaizumi Polymorphonuclear Leukocytes May Impair Endothelial Function: Results of Crossover Randomized Study of Lipid-Lowering Therapies Arterioscler Thromb Vasc Biol, June 1, 2005; 25(6): 1262 - 1267. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Touyz Reactive Oxygen Species, Vascular Oxidative Stress, and Redox Signaling in Hypertension: What Is the Clinical Significance? Hypertension, September 1, 2004; 44(3): 248 - 252. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Prasad C-Reactive Protein Increases Oxygen Radical Generation by Neutrophils Journal of Cardiovascular Pharmacology and Therapeutics, July 1, 2004; 9(3): 203 - 209. [Abstract] [PDF]
|
|
|
|
 |
|
 K. Yasunari, K. Maeda, T. Watanabe, M. Nakamura, J. Yoshikawa, and A. Asada Comparative effects of valsartan versus amlodipine on left ventricular mass and reactive oxygen species formation by monocytes in hypertensive patients with left ventricular hypertrophy J. Am. Coll. Cardiol., June 2, 2004; 43(11): 2116 - 2123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-T. Chien, W.-T. Chang, H.-W. Chen, T.-D. Wang, S.-Y. Liou, T.-J. Chen, Y.-L. Chang, Y.-T. Lee, and S.-M. Hsu Ascorbate Supplement Reduces Oxidative Stress in Dyslipidemic Patients Undergoing Apheresis Arterioscler Thromb Vasc Biol, June 1, 2004; 24(6): 1111 - 1117. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Chon, C. A.J.M. Gaillard, B. B. van der Meijden, H. M. Dijstelbloem, R. J. Kraaijenhagen, D. van Leenen, F. C.P. Holstege, J. A. Joles, H. A.R. Bluyssen, H. A. Koomans, et al. Broadly Altered Gene Expression in Blood Leukocytes in Essential Hypertension Is Absent During Treatment Hypertension, May 1, 2004; 43(5): 947 - 951. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. W. Cohen, S. M. Hailpern, and M. H. Alderman Glucose-Cholesterol Interaction Magnifies Coronary Heart Disease Risk for Hypertensive Patients Hypertension, May 1, 2004; 43(5): 983 - 987. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. J. Blake, N. Rifai, J. E. Buring, and P. M Ridker Blood Pressure, C-Reactive Protein, and Risk of Future Cardiovascular Events Circulation, December 16, 2003; 108(24): 2993 - 2999. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Tepel Oxidative stress: does it play a role in the genesis of essential hypertension and hypertension of uraemia? Nephrol. Dial. Transplant., August 1, 2003; 18(8): 1439 - 1442. [Full Text] [PDF]
|
|
|
|
|
|
L. A. Calo, E. Pagnin, P. A. Davis, M. Sartori, and A. Semplicini Oxidative stress-related factors in Bartter's and Gitelman's syndromes: relevance for angiotensin II signalling Nephrol. Dial. Transplant., August 1, 2003; 18(8): 1518 - 1525. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Tepel Oxidative stress: does it play a role in the genesis of essential hypertension and hypertension of uraemia? Nephrol. Dial. Transplant., August 1, 2003; 18(88): 1439 - 1442. [Full Text]
|
|
|
|
|
|
L. A. Calo, E. Pagnin, P. A. Davis, M. Sartori, and A. Semplicini Oxidative stress-related factors in Bartter's and Gitelman's syndromes: relevance for angiotensin II signalling Nephrol. Dial. Transplant., August 1, 2003; 18(88): 1518 - 1525. [Abstract] [Full Text]
|
|
|
|
 |
|
 G. Engstrom, B. Hedblad, L. Stavenow, P. Lind, L. Janzon, and F. Lindgarde Inflammation-Sensitive Plasma Proteins Are Associated With Future Weight Gain Diabetes, August 1, 2003; 52(8): 2097 - 2101. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Katsuki, Y. Sumida, H. Urakawa, E. C. Gabazza, N. Maruyama, K. Morioka, N. Kitagawa, Y. Hori, K. Nakatani, Y. Yano, et al. Increased Oxidative Stress Is Associated With Elevated Plasma Levels of Adrenomedullin in Hypertensive Patients With Type 2 Diabetes Diabetes Care, May 1, 2003; 26(5): 1642 - 1643. [Full Text] [PDF]
|
|
|
|
|
|
J. Redon, M. R. Oliva, C. Tormos, V. Giner, J. Chaves, A. Iradi, and G. T. Saez Antioxidant Activities and Oxidative Stress Byproducts in Human Hypertension Hypertension, May 1, 2003; 41(5): 1096 - 1101. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||