Abstract
Background
The p66shc protein has been shown to control cellular responses to oxidative stress, being involved in atherosclerosis in animal models. However, the relationship between the p66shc gene expression levels and coronary artery disease (CAD) in humans remains unknown. In this study, we examined whether the p66shc gene expression in peripheral blood monocytes (PBMs) was increased in patients with CAD, compared with age‐ and sex‐matched subjects without CAD.
Hypothesis
We hypothesize that the p66shc gene expression level in PBMs is increased in patients with CAD.
Methods
Forty consecutive Japanese subjects who underwent coronary angiography for suspected CAD were enrolled in this study. The p66shc gene expression levels in PBMs were quantitatively measured by real‐time reverse transcription‐polymerase chain reactions. Uni‐ and multivariate analyses were applied for the correlates of CAD. CAD was diagnosed if there was > 75% obstruction of at least 1 major coronary artery or a history of percutaneous coronary intervention.
Results
There were no significant differences of blood chemistries and clinical characteristics between the patients with and without CAD, except the number of subjects who were on hypertension medication. The p66shc gene expression levels in PBMs were significantly higher in CAD patients compared with non‐CAD subjects. Multiple stepwise regression analysis revealed that the p66shc gene expression levels and hypertension medication were independently related to CAD (R2=0.287). Further, the p66shc gene expres‐ sion levels were significantly increased (P < 0.05) in proportion to the number of diseased vessels.
Conclusions
The present study is the first demonstration that increased the p66shc gene expression in PBMs is independently associated with CAD in Japanese subjects. The p66shc gene expression level in PBMs may be a novel biomarker of CAD in humans. Copyright © 2010 Wiley Periodicals, Inc.
This work was supported in part by Grants of Collaboration with Venture Companies Project from the Ministry of Education, Culture, Sports, Science and Technology, Japan (S. Yamagishi). The authors have no other funding, financial relationships, or conflicts of interest to disclose.
Introduction
There is accumulating evidence, ranging from in vitro experiments to pathologic analysis to epidemiologic studies, that oxidative stress plays an important role in the pathogenesis of atherosclerosis.1, 2, 3, 4 Further, recently, the p66shc protein has been shown to control cellular responses to oxidative stress, being involved in atherosclerosis in animal models.5, 6, 7, 8, 9 Indeed, deletion of the p66shc gene not only protects against age‐related endothelial dysfunction, an initial step of atherosclerosis,7 but also reduces systemic and tissue oxidative stress, vascular cell apoptosis, and early atherogenesis in mice fed a high‐fat diet.8 In addition, the p66shc gene deletion has also been found to confer vascular protection in advanced atherosclerosis in hypercholesterolemic apolipoprotein E knockout mice.9 These observations suggest that gene expression levels of the p66shc could regulate atherosclerosis in humans. However, as far as we know, there has been no report to have examined the correlation of gene expression levels of the p66shc with atherosclerosis in humans. Therefore, in this study, we investigated whether the p66shc gene expression in peripheral blood monocytes (PBMs) was increased in patients with coronary artery disease (CAD) compared with age‐ and sex‐matched subjects without CAD.
Research Design and Methods Subjects
The study population consisted of 40 consecutive Japanese subjects (29 males and 11 females) who underwent coronary angiography for suspected CAD from January 2009 to June 2009 at the Division of Cardio‐Vascular Medicine, Kurume University Hospital. The numbers of patients who received medication for diabetes mellitus (DM), hypertension (HT), and hyperlipidemia (HL) were 17, 32, and 24, respectively. We excluded any patients with neoplastic disorders or collagen disease, and those who had recent (<3 months) acute coronary syndromes and stroke, a history of coronary artery bypass graft surgery, and any acute infection. The Ethical Committee of Kurume University approved this study. All participants gave informed consent.
Data Collection
Height and weight were measured, and body mass index (weight in kilograms divided by height in meters squared) was calculated as an index of the presence or absence of obesity. Blood pressure was measured in the sitting position using an upright standard sphygmomanometer. Vigorous physical activity was avoided for at least 30 minutes before blood pressure measurement.
Blood was drawn from the antecubital vein after admission to the hospital for determination of lipids (total cholesterol, high‐density lipoprotein cholesterol, low‐density lipoprotein cholesterol, and triglycerides), plasma glucose, glycosylated hemoglobin (HbA1c), blood urea nitrogen, creatinine, uric acid, aspartate aminotransferase, alanine aminotransferase, and γ ‐ glutamyl transpeptidase. Blood chemistries were measured at our laboratory, using an enzyme‐linked immunosorbent assay or an enzymatic assay method as described previously.10 Glomerular filtration rate was estimated with the Modification of Diet in Renal Disease study equation modified with a Japanese coefficient.11
Severity of coronary artery stenoses were evaluated by more than 2 experienced interventional cardiologists blinded to the p66shc data, using quantitative coronary angiography. CAD was diagnosed if there was > 75% obstruction of at least 1 major coronary artery (right, left anterior descending, or left circumflex coronary artery) or a history of percutaneous coronary intervention. According to the number of diseased major coronary arteries (0, 1, 2, or 3), the p66shc gene expression levels in PBMs were stratified. When the lesion was detected in the left main coronary artery, the number of diseased vessels was considered 2.
Peripheral Blood Monocyte Preparation
Monocytes were isolated from peripheral blood as described previously.12 Briefly, 14‐mL anticoagulated blood samples were taken from each subject. Buffy coats were separated by Ficoll‐Paque PLUS gradient (GE Healthcare Bio‐Sciences AB, Uppsala, Sweden). Mononuclear cells obtained were suspended in Roswell Park Memorial Institute tissue culture medium (RPMI 1640; Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Invitrogen Corp., Carlsbad, CA), and placed for 12 hours at 37 °C. After removing the medium and rinsing twice with phosphate‐buffered saline, cells adherent on the flasks were collected with a cell scraper. We confirmed that most of the adherent cells (>99%) were monocytes with Diff‐Quik staining (Sysmex, Kobe, Japan).
Real‐Time Reverse Transcription‐Polymerase Chain Reactions
Total RNA was extracted from PBMs with the TRIzol Plus RNA Purification Kit (Invitrogen) according to the manufacturer's instructions. Quantitative real‐time reverse transcription‐polymerase chain reactions were performed using Assay‐on‐Demand and TaqMan 5 fluorogenic nuclease chemistry (Applied Biosystems, Foster City, CA) according to the supplier's recommendation. The IDs of primers for human the p66shc and 18S genes were Hs00427539_m1 and Hs99999901_s1, respectively.
Statistical Methods
Data were expressed as mean ± SD. To compare the clinical parameters between patients with CAD and without CAD, we used the Student t test or χ2 test. The medications for HT, DM, and HL were coded as dummy variables. Univariate analysis was performed for correlates of CAD. To determine independent correlates of CAD, multiple stepwise regression analysis was performed. Statistical significance was defined as P < 0.05. All statistical analyses were performed with the SAS system (SAS Institute, Cary, NC).
Results
Backgrounds of the subjects are presented in Table 1. There were no significant differences of blood chemistries and clinical characteristics between the patients with and without CAD, except the number of subjects who were on HT medication and total cholesterol levels. As shown in Table 1 and Figure 1, the p66shc gene expression levels in PBMs were significantly higher in CAD patients compared with non‐CAD subjects. Table 2 shows results of univariate analysis for correlates of CAD. Parameters statistically and significantly related to CAD were the p66shc gene expression levels (P = 0.01) and HT medication (P = 0.01). Because the p66shc levels and HT medication significant parameters could be closely correlated with each other, multiple stepwise regression analysis was performed. Finally, the p66shc gene expression levels (P = 0.026) and HT medication (P = 0.006) remained significant and were independently related to CAD (R2=0.287). Further, the p66shc gene expression levels in PBMs were significantly increased (P < 0.05 by analysis of variance [ANOVA]) in proportion to the number of diseased vessels (Fig. 1).
Table 1.
Clinical Characteristics of the Patients
Characteristics | CAD(−)n=18 | CAD(+)n=22 | P Value |
---|---|---|---|
Age (y) | 69.7±9.5 | 71.7±6.5 | 0.442 |
Sex (m/f) | 11/7 | 18/4 | 0.173 |
BMI (kg/m2) | 23.8±3.6 | 23.9±3.2 | 0.926 |
p66shc (arbitrary units) | 0.87±0.19 | 1.16±0.28 | <0.001 |
AST (IU/L) | 26.7±7.2 | 25.1±8.6 | 0.524 |
ALT (IU/L) | 20.2±7.5 | 20.8±9.3 | 0.824 |
γ‐GTP (IU/L) | 34.3±26.2 | 36.6±20.9 | 0.758 |
Total cholesterol (mg/dL) | 187.4±31.8 | 162.1±35.6 | 0.0247 |
Triglycerides (mg/dL) | 133.2±60.5 | 125.1±74.5 | 0.714 |
HDL‐cholesterol (mg/dL) | 55.1±19.6 | 48.4±10.9 | 0.181 |
LDL‐cholesterol (mg/dL) | 114.8±26.7 | 102.0±31.7 | 0.186 |
Creatinine (mg/dL) | 0.80±0.26 | 0.94±0.30 | 0.135 |
eGFR (mL/min/1.73 m2) | 72.0±21.2 | 63.1±17.3 | 0.149 |
BUN (mg/dL) | 18±9.3 | 17.8±4.9 | 0.821 |
Glucose (mg/dL) | 147.1±48.3 | 141.7±56.5 | 0.751 |
HbA1c (%) | 6.0±1.0 | 6.3±1.1 | 0.548 |
SBP (mm Hg) | 129.4±25.8 | 129.5±22.5 | 0.999 |
DBP (mm Hg) | 71.8±15.7 | 68.2±12.3 | 0.415 |
Uric acid (mg/dL) | 5.7±1.8 | 5.9±1.9 | 0.751 |
No. | |||
DM medication | 5 | 12 | 0.088 |
HT medication | 11 | 21 | 0.010 |
HL medication | 9 | 15 | 0.243 |
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; BUN, blood urea nitrogen; CAD, coronary artery disease; DBP, diastolic blood pressure; DM, diabetes mellitus; eGFR, estimated glomerular filtration rate; f, female; γ‐GTP, γ‐glutamyl transpeptidase; HbA1c, glycosylated hemoglobin; HDL, high‐density lipoprotein; HL, hyperlipidemia; HT, hypertension; LDL, low‐density lipoprotein; m, male; min, minute; n, number of patients; SBP, systolic blood pressure; y, years.
a Values are mean±SD or percentage, unless indicated otherwise
Figure 1.
The p66shc gene expression levels in PBMs stratified by the number of diseased vessels
Table 2.
Correlates of CAD by Multiple Stepwise Logistic Regression Analysis
Factors | Univariatea | Multivariateb | |||
---|---|---|---|---|---|
β | P | β | F | P | |
Age (y) | 0.005 | 0.630 | |||
Sex | 0.229 | 0.210 | |||
Height (cm) | 0.011 | 0.199 | |||
Weight (kg) | 0.008 | 0.278 | |||
BMI (kg/m2) | 0.011 | 0.680 | |||
p66shc (arbitrary units) | 0.707 | 0.010 | 0.573 | 5.41 | 0.026 |
AST (IU/L) | ‐0.011 | 0.310 | |||
ALT (IU/L) | ‐0.001 | 0.365 | |||
γ‐GTP (IU/L) | 0.004 | 0.289 | |||
Total cholesterol (mg/dL) | ‐0.002 | 0.353 | |||
Triglycerides (mg/dL) | 0.000 | 0.887 | |||
HDL‐cholesterol (mg/dL) | ‐0.006 | 0.252 | |||
LDL‐cholesterol (mg/dL) | ‐0.004 | 0.161 | |||
Creatinine (mg/dL) | 0.429 | 0.130 | |||
eGFR (mL/min/1.73 m2) | ‐0.006 | 0.152 | |||
BUN (mg/dL) | ‐0.002 | 0.864 | |||
Glucose (mg/dL) | ‐0.001 | 0.780 | |||
HbA1c (%) | 0.061 | 0.436 | |||
SBP (mmHg) | 0.000 | 0.859 | |||
DBP (mmHg) | ‐0.004 | 0.490 | |||
Uric acid (mg/dL) | 0.008 | 0.865 | |||
HT medication | 0.508 | 0.010 | 0.531 | 8.48 | 0.006 |
HL medication | 0.268 | 0.117 | |||
DM medication | 0.278 | 0.094 |
Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; BUN, blood urea nitrogen; CAD, coronary artery disease; DBP, diastolic blood pressure; DM, diabetes mellitus; eGFR, estimated glomerular filtration rate; f, female; γ‐GTP, γ‐glutamyl transpeptidase; HbA1c, glycosylated hemoglobin; HDL, high‐density lipoprotein; HL, hyperlipidemia; HT, hypertension; LDL, low‐density lipoprotein; m, male; min, minute; SBP, systolic blood pressure; y, years.
Univariate coefficients.
A multiple stepwise logistic regression analysis was performed. β: Regression coefficients. Female=0, male=1
Discussion
In this study, we found for the first time that the p66shc gene expression levels in PBMs were significantly higher in CAD patients compared with non‐CAD subjects and that increased the p66shc gene expression was independently associated with CAD. Since the p66shc gene expression levels were significantly increased in proportion to the number of diseased vessels, our present findings suggest that the p66shc gene expression level in PBMs may be a novel biomarker of CAD in humans.
Recently, several papers have shown the active involvement of the p66shc adaptor protein in vascular injury in animal models.5, 6, 7, 8, 9 Indeed, overexpression of the p66shc gene has been shown to reduce nitric oxide generation in endothelial cells, whereas downregulation or deletion of the p66shc gene improves impaired endothelial cell‐dependent vasodilation, a key step for atherosclerosis.5, 7 Further, both early and advanced atherosclerotic lesion formations were suppressed in the p66shc knockout mice.8, 9 These findings led us to speculate that the p66shc gene expression could be associated with atherosclerosis in humans. Further, there is accumulating evidence that monocytes/macrophages play a central role in the pathogenesis of atherosclerosis in human.13, 14 In addition, it is a simple and time‐saving task to isolate monocytes from peripheral blood and analyze gene expression in PBMs. These are reasons why the p66shc gene expression level in PBMs was chosen in this study as a marker of interest.
As far as we know, there is only one paper which showed that the levels of the p66shc gene expression in PBMs were significantly increased in patients with type 2 DM compared with controls, and the levels were correlated to total plasma 8‐isoprostane levels, a marker of oxidative stress.15 However, in this study, serum level of 8‐hydroxy‐2′‐ deoxyguanosine (8‐OHdG), another marker of oxidative stress, was not higher in CAD patients than in non‐CAD subjects (0.095 ± 0.036 vs 0.086 ± 0.074 ng/mL) and was not correlated with the p66shc gene expression levels in PBMs (data not shown). The subject population and the presence or absence of medication could account for the discrepant results between theirs and ours. Anyway, although we did not clarify the molecular mechanism for the elevation of the p66shc gene expression levels in PBMs in our CAD patients, the present findings suggest that the p66shc gene expression in PBMs may represent a useful marker of vascular injury in high‐risk patients such as those with DM and/or CAD.
It is noted that traditional risk factors for CAD such as dyslipidemia, hyperglycemia, and HT were not associated with CAD in our subjects, probably because many patients were on medications. The association of HT medication with CAD simply indicates hypertension as a risk factor for CAD.
Limitations
The present study is a small number and a cross‐sectional study and therefore does not elucidate the causal relationships between the p66shc gene expression levels in PBMs and the presence of CAD. A further longitudinal study with a relatively large number of patients is needed to clarify the issue of whether the p66shc gene expression level in PBMs could be a novel marker for CAD and whether reduction of the p66shc gene expression could prevent future cardiovascular events in high‐risk patients.
REFERENCES
- 1. Briasoulis A, Tousoulis D, Antoniades C, et al. The oxidative stress menace to coronary vasculature: any place for antioxidants? Curr Pharm Des 2009; 15: 3078–3090. [DOI] [PubMed] [Google Scholar]
- 2. Victor VM, Rocha M, Solá E, et al. Oxidative stress, endothelial dysfunction and atherosclerosis. Curr Pharm Des 2009; 15: 2988–3002. [DOI] [PubMed] [Google Scholar]
- 3. Yamagishi S, Nakamura K, Matsui T. Role of oxidative stress in the development of vascular injury and its therapeutic intervention by nifedipine. Curr Med Chem 2008; 15: 172–177. [DOI] [PubMed] [Google Scholar]
- 4. Antonopoulos AS, Antoniades C, Tousoulis D, et al. Novel therapeutic strategies targeting vascular redox in human atherosclerosis. Recent Pat Cardiovasc Drug Discov 2009; 4: 76–87. [DOI] [PubMed] [Google Scholar]
- 5. Francia P, Cosentino F, Schiavoni M, et al. p66(Shc) protein, oxidative stress, and cardiovascular complications of diabetes: the missing link. J Mol Med 2009; 87: 885–891. [DOI] [PubMed] [Google Scholar]
- 6. Pellegrini M, Baldari CT. Apoptosis and oxidative stress‐related diseases: the p66Shc connection. Curr Mol Med 2009; 9: 392–398. [DOI] [PubMed] [Google Scholar]
- 7. Francia P, delli Gatti C, Bachschmid M, et al. Deletion of p66shc gene protects against age‐related endothelial dysfunction. Circulation 2004; 110: 2889–2895. [DOI] [PubMed] [Google Scholar]
- 8. Napoli C, Martin‐Padura I, de Nigris F, et al. Deletion of the p66Shc longevity gene reduces systemic and tissue oxidative stress, vascular cell apoptosis, and early atherogenesis in mice fed a high‐fat diet. Proc Natl Acad Sci U S A 2003; 100: 2112–2116. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Martin‐Padura I, de Nigris F, Migliaccio E, et al. p66Shc deletion confers vascular protection in advanced atherosclerosis in hypercholesterolemic apolipoprotein E knockout mice. Endothelium 2008; 15: 276–287. [DOI] [PubMed] [Google Scholar]
- 10. Tahara N, Kai H, Yamagishi S, et al. Vascular inflammation evaluated by [18F]‐fluorodeoxyglucose positron emission tomography is associated with the metabolic syndrome. J Am Coll Cardiol 2007; 49: 1533–1539. [DOI] [PubMed] [Google Scholar]
- 11. Imai E, Horio M, Nitta K, et al. Estimation of glomerular filtration rate by the MDRD study equation modified for Japanese patients with chronic kidney disease. Clin Exp Nephrol 2007; 11: 41–50. [DOI] [PubMed] [Google Scholar]
- 12. Miura J, Uchigata Y, Yamamoto Y, et al. AGE down‐regulation of monocyte RAGE expression and its association with diabetic complications in type 1 diabetes. J Diabetes Complications 2004; 18: 53–59. [DOI] [PubMed] [Google Scholar]
- 13. Wilson HM, Barker RN, Erwig LP. Macrophages: promising targets for the treatment of atherosclerosis. Curr Vasc Pharmacol 2009; 7: 234–243. [DOI] [PubMed] [Google Scholar]
- 14. Saha P, Modarai B, Humphries J, et al. The monocyte/macrophage as a therapeutic target in atherosclerosis. Curr Opin Pharmacol 2009; 9: 109–118. [DOI] [PubMed] [Google Scholar]
- 15. Pagnin E, Fadini G, de Toni R, Tiengo A, Calò L, Avogaro A. Diabetes induces p66shc gene expression in human peripheral blood mononuclear cells: relationship to oxidative stress. J Clin Endocrinol Metab 2005; 90: 1130–1136. [DOI] [PubMed] [Google Scholar]