Heart Vessels DOI 10.1007/s00380-014-0497-4

ORIGINAL ARTICLE

The expression of p66shc in peripheral blood monocytes is increased in patients with coronary heart disease and correlated with endothelium-dependent vasodilatation Qin Miao • Qiong Wang • Lini Dong • Yanjiao Wang • Yi Tan • Xiangyu Zhang

Received: 14 November 2013 / Accepted: 14 March 2014 Ó Springer Japan 2014

Abstract The objective of this study is to detect the p66shc mRNA and protein expression of the peripheral blood monocytes (PBMs) in coronary heart disease patients (CHD) and controls, to evaluate the correlation between the expression of p66shc mRNA in the PBMs and endothelium-dependent vasodilatation. This study included 78 coronary angiography-documented CHD patients (CHD group) and 38 non-CHD controls (control group). The p66shc mRNA and protein levels were determined by quantitative real-time PCR and western blotting. The flowmediated dilatation (FMD, endothelium-dependent), nitroglycerine-induced dilatation (NID, endothelium-independent) and carotid intimal medial thickness (CIMT) were detected using high-resolution ultrasound. The p66shc mRNA and the protein expression levels in the PBMs were significantly higher in the CHD group compared with the control group (p = 0.007 and 0.001). The FMD (p \ 0.001) and NID (p = 0.013) were significantly lower and the CIMT (p = 0.007) was significantly thicker in the CHD patients than in the controls. In the univariate analysis, the expression of the p66shc mRNA in the PBMs was

Q. Miao  Q. Wang  L. Dong  Y. Wang  X. Zhang (&) Department of Geriatrics, Second Xiangya Hospital of Central South University, No. 139, Middle Renmin Road, Changsha 410011, Hunan, People’s Republic of China e-mail: [email protected] Present Address: Q. Miao Department of Geriatrics, Changsha Central hospital, Changsha 410001, Hunan, People’s Republic of China Y. Tan Department of Ultrasound, Second Xiangya Hospital of Central South University, Changsha 410011, Hunan, People’s Republic of China

significantly positively correlated with the serum LDL-C and homocysteine levels and the CIMT and was inversely correlated with the FMD and the NID (all p \ 0.001). In the multiple linear regression analysis, the FMD (p \ 0.001), LDL-C (p = 0.002) and homocysteine levels (p = 0.002) remained independently correlated with the p66shc mRNA expression. These findings highlight a pivotal role for the expression of p66shc in CHD and endothelial dysfunction, which might represent a molecular target to prevent endothelial dysfunction-related disease. Keywords p66shc  Endothelium-dependent vasodilatation  Coronary heart disease  Peripheral blood monocytes  Homocysteine

Introduction Increased production of reactive oxygen species (ROS) is the common dominator of vascular aging, endothelial dysfunction and atherosclerosis. Oxidative stress caused by ROS has been widely accepted as the major pathogenetic factor of vascular disease and a major determinant of endothelial dysfunction [1, 2]. The vascular endothelium plays an essential role in the regulation of blood flow, inhibition of platelet aggregation and adhesion and prevention of monocytes from adhering to the vascular wall. Endothelial dysfunction, which is characterized by impaired endothelium-dependent vasodilatation, has a close correlation with atherosclerosis and coronary artery disease and represents an early step in the pathogenesis of cardiovascular diseases [3, 4]. ROS are generated by different intracellular molecular pathways. Recently, accumulating evidence demonstrated that p66shc is a key regulator for oxidative stress. P66shc

123

Heart Vessels

belongs to the shcA family of adaptor proteins, which includes three isoforms of p46shc, p52shc and p66shc. Among them, p66shc is the only isoform that acts as a redox enzyme implicated in mitochondrial ROS generation, mediating oxidative stress in many cell types and tissues, and translating the oxidative signals into apoptosis. p66shcdeficient mice have an enhanced resistance to oxidative stress and a 30 % increase in lifespan [5]. The known role of p66shc in ROS generation is relevant to its involvement in atherosclerosis and cardiovascular disease. It has been demonstrated that genetic deletion of p66shc can decrease superoxide production in macrophages [6]; reduce systemic and tissue oxidative stress, vascular cell apoptosis and early atherogenesis in mice fed a high-fat diet [7]; and reduce the content of macrophage-derived foam cells and apoptotic vascular cells [8]. Atherosclerosis is closely correlated with endothelial dysfunction. Overexpression of p66shc has been shown to inhibit eNOS-dependent NO production [9], whereas down-regulation or deletion of p66shc improves endothelium-dependent vasorelaxation and prevents agerelated or hyperglycemia-induced endothelial dysfunction and oxidative stress [10, 11]. These findings suggest that gene expression levels of p66shc could regulate atherosclerosis and foam cell formation in atherogenesis as well as endothelial dysfunction. Despite the growing body of evidence implicating the critical role of p66shc in the pathophysiology of cardiovascular diseases in animal models and ex vivo studies, there is limited information in humans. Three recent studies reported that p66shc mRNA levels were increased in the peripheral blood mononuclear cells or monocytes (PBMs) of diabetes mellitus [12], coronary artery disease (CAD) [13] and acute coronary syndrome (ACS) patients [14]. The role of p66shc in the endothelial dysfunction of coronary heart disease (CHD) patients has not been investigated. The aims of the current study were as follows: (1) to detect the p66shc mRNA and protein expression in PBMs of CHD patients and controls; (2) to use high-resolution ultrasound to test the endothelium-dependent and independent vascular dilatation and the carotid intima media thickness (CIMT) of the study subjects and to evaluate whether they correlated with the expression of p66shc mRNA in the PBMs; and (3) to determine the relationship between the p66shc mRNA expression in PBMs and other CHD risk factors.

Materials and methods Participants This study included 78 CHD patients (CHD group) and 38 non-CHD controls (control group). All of the subjects

123

underwent coronary angiography. The coronary angiograms were read by two experienced technicians blinded to the patient identity, the clinical diagnosis and the study. The CHD group included stable CAD (n = 15) patients and ACS patients (n = 63) in stable conditions. A diagnosis of CAD indicated no less than 75 % luminal diameter stenosis in the left main, left anterior descending, left circumflex or right coronary artery. A diagnosis of ACS was documented with electrocardiography, angiographic findings and laboratory markers of myocardial troponin T and creatine kinase. The extent of CAD was quantified according to the number of the three major coronary arteries with stenosis [75 % (one-, two- and three-vessel disease groups), with the left main artery counting as two vessels. Seventeen subjects had one-, 25 subjects had two- and 36 subjects had three-vessel disease in the CHD group. The patients were excluded if they suffered from hepatic or renal failure, rheumatic heart disease, cardiomyopathy, acute or chronic infection, malignancy, autoimmune disease or connective tissue disease. The ACS patients in unstable and severe conditions were excluded. All the data of these participants were recorded and systematically entered into a Microsoft Excel chart. This study was approved by the institutional ethics committee of the 2nd Xiangya Hospital of Central South University. All of the subjects provided written informed consent. Blood collection and biochemical analyses Venous blood was drawn from all of the subjects under standardized conditions after a 12 h fast before coronary angiography was performed. The serum samples were collected by centrifugation at 4,000 rpm for 10 min. The serum triglycerides (TG), total cholesterol (TC), highdensity lipoprotein cholesterol (HDL-C), high-sensitivity C-reactive protein (hs-CRP), fasting blood glucose levels, the liver and renal functions were determined at the central chemistry laboratory of our hospital using standard automated enzymatic methods on a Hitachi 912 automated analyzer with reagents from Kamiya Biomedical Company. The LDL-C level was calculated using the Friedwald formula. The homocysteine (Hcy) level was measured using the enzymatic cycling method. Peripheral blood monocyte preparation The anticoagulated blood was centrifuged at 2,000 rpm for 20 min at 20 °C. The blood cells were diluted with phosphate-buffered saline (PBS) and carefully layered on Ficoll-Paque PLUS (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) in a polypropylene conical-bottomed tube and centrifuged at 2,000 rpm for 30 min. The turbid white layer containing the mononuclear blood cells was transferred to a clean tube and washed twice with PBS at

Heart Vessels

1,500 rpm for 10 min; the supernatant was removed after washing. The obtained mononuclear cells were suspended in RPMI 1640 culture medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10 % heat-inactivated fetal bovine serum (Invitrogen Corp., Carlsbad, CA, USA) allowing the monocytes to adhere to the flasks overnight at 37 °C, 5 % CO2. Then the medium was removed and the cells were rinsed twice with PBS, the cells adherent on the flasks were collected and ready for the subsequent experiments. The purity of CD14-positive monocytes was assessed by flow cytometry. RNA isolation and real-time RT-PCR All the samples of CHD patients and the control groups were determined for the p66shc mRNA expression. Total RNA was extracted from the prepared PBMs by the TRIzol isolation method (Invitrogen). The concentration of RNA samples was measured with a spectrophotometer. Total RNA (1 lg) was used for making the cDNA with a PrimeScriptTM RT reagent kit with a gDNA Eraser (TaKaRa code: DRR047A). The quantitative real-time PCR was performed using the SYBRTM Premix Ex TaqTM II Kit (TaKaRa code: DRR081A). The specific primers (synthesized by TaKaRa) for p66shc were forward: 50 TCCTCCAGGACATGAACAAGCTGA-30 ; reverse: 50 TGGGCTTATTGACAAAGCTCCCGT-30 . All of the PCRs were performed on a 7900 Fast Real-Time PCR System (Applied BiosystemsTM) under the following conditions: 95 °C for 30 min, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Each sample was performed in triplicate and normalized to 18S. The quantification was performed using the 2DDCt method. Western blotting We randomly selected 12 CHD and 12 control subjects and determined their p66shc protein expression in PBMs using western blot analysis. The protein concentration was measured by the bicinchoninic acid (BCA) method. Thirty micrograms of the sample protein was separated by 12 % polyacrylamide-sodium dodecyl sulfate gels (SDS-PAGE) and transferred to a nitrocellulose membrane. Western blotting was performed using the monoclonal antibody for p66shc (1:500, 4 °C overnight; Abcam, catalog# ab33770, USA) and the anti-rabbit IgG secondary antibody (1:40,000, KPL, catalog# sc-04-1506, USA). We used GAPDH as the loading control. The immunoblots were scanned, and the bands were quantified using the Kodak 1D image analysis system. The densitometric values were normalized to GAPDH to control for gel loading.

Ultrasound image analysis The high-resolution ultrasound imaging was conducted by an experienced radiologist, unaware of the patient’s history or laboratory findings using GE VIVI 7 (USA) with a 10.0MHz linear array probe. The flow-mediated dilatation (FMD) and the nitroglycerine-induced dilatation (NID) responses were measured as endothelium-dependent and endothelium-independent vasodilatation, respectively. They were performed as we previously described [15]. After fasting for 12 h and discontinuation of long-acting vasoactive medications for 24 h, the subjects were examined in a resting supine state in a temperature-controlled room. The brachial artery was imaged at a location 3–7 cm above the antecubital crease of the right arm. The baseline measurements of the brachial artery diameter were recorded. A blood pressure cuff was inflated on the proximal portion of the arm to 250–300 mmHg, and the brachial artery was scanned continuously 30 s before and 90 s after the cuff deflation. When the brachial artery returned to the baseline level, 0.5 mg of nitroglycerin was administered sublingually, and the brachial artery was imaged after *4 min. All of the vasodilatation measurements were performed at end diastole incident with the R wave on a continuously recorded electrocardiogram. An average of three consecutive measurements, each taken 10 min apart, was taken for each subject. The flow-mediated and nitroglycerine-induced dilation were calculated as the percent change in the brachial artery diameter (vasodilatation), which was determined by dividing the difference from the baseline diameter by the baseline value. CIMT was measured at the distal walls of right and left common carotid artery in three contiguous sites of 1-cm intervals, from the beginning of the common carotid bulb. It was identified as the region between the lumen–intima interface and the media–adventitia interface. The patients were examined in the supine position with the head turned 45° contralateral to the side of scanning. A total of six measurements of both sides per patient were averaged. Statistical analysis The data were analyzed by a medical statistician using SPSS (18.0). The descriptive data were expressed as the mean value ± standard deviation (SD). After testing for a normal distribution of the variables, Student’s two-tailed t test was used for the comparisons between the paired and un-paired data, and one-way ANOVA was used for the continuous variables, followed by Scheffe’s test for the multiple comparisons. The univariate and multivariate analyses of the effects factor on the p66shc mRNA

123

Heart Vessels

expression were performed with linear regression analysis. A two-tailed p value \0.05 was considered to be statistically significant.

Results The clinical characteristics of the CHD and control groups are shown in Table 1. The blood levels of hs-CRP, LDL-C and Hcy were significantly higher in the CHD group than in the control group. There were no significant differences

Table 1 Clinical characteristics of the CHD patients and controls CHD (n = 78)

Control (n = 38)

p value

Age (years)

57.0 ± 7.0

54.6 ± 7.6

Gender (male %)

53 (68.42 %)

24 (63.89 %)

0.677

BMI (kg/m2)

23.96 ± 2.68

22.98 ± 2.55

0.114

0.153

SBP (mmHg)

127.07 ± 15.61

124.68 ± 10.80

0.073

DBP (mmHg)

78.62 ± 9.63

76.94 ± 7.38

0.165

Glucose (mmol/L)

5.47 ± 1.46

5.75 ± 1.46

0.420

Smoking Diabetes

52 (66.66 %) 18 (23.68 %)

23 (60.53 %) 6 (16.67 %)

0.540 0.467

Hypertension

43 (55.26 %)

20 (52.78 %)

0.844

ACEI

21 (26.32 %)

10 (25 %)

1.000

Statin

16 (21.05 %)

8 (22.22 %)

1.000

ALT (u/L)

37.65 ± 29.08

28.10 ± 14.69

0.086

AST (u/L)

27.11 ± 16.24

23.08 ± 8.93

0.213

TG (mmol/L)

2.09 ± 1.03

2.02 ± 0.56

0.746

TC (mmol/L)

5.24 ± 1.28

5.12 ± 0.95

0.072

HDL-C (mmol/L)

0.90 ± 0.28

1.01 ± 0.24

0.065

LDL-C (mmol/L)

3.39 ± 1.28

3.16 ± 0.85

0.044

BUN (mmol/L)

5.72 ± 1.52

5.09 ± 1.37

0.065

CR (lmol/L)

84.94 ± 17.98

76.78 ± 20.50

0.072

Uric acid (lmol/L)

364.95 ± 110.70

344.93 ± 106.55

0.431

Hs-CRP (mg/L)

8.83.47 ± 15.74

2.43 ± 1.54

0.039

Hcy (lmol/L) Baseline vessel diameter (mm)

16.94 ± 8.70 3.85 ± 0.58

13.63 ± 3.63 4.02 ± 0.72

0.017 0.439

FMD (%)

8.72 ± 5.03

13.74 ± 2.31

\0.001

NID (%)

11.80 ± 5.15

15.36 ± 3.28

0.013

CIMT (mm)

0.82 ± 0.26

0.63 ± 0.16

0.007

P66shc mRNA

1.36 ± 0.74

0.94 ± 0.51

0.007

P66shc protein

1.40 ± 0.101

1.03 ± 0.076

0.001

BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, ACEI angiotensin-converting enzyme inhibitor, ALT alanine aminotransferase, AST aspartate aminotransferase, TG triglyceride, TC total cholesterol, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, BUN blood urea nitrogen, CR serum creatinine, Hs-CRP high-sensitive C-reactive protein, Hcy homocysteine, FMD flow-mediated dilatation, NID nitroglycerine-induced dilatation, CIMT carotid intimal medial thickness

123

in the other blood chemistries and in the clinical characteristics between the two groups. There were no significant differences in the baseline brachial artery diameters in the CHD group and the control group. The FMD and NID were significantly lower in the CHD patients than in the controls (Fig. 1). There was significant difference between FMD and NID in CHD patients (p = 0.039) and no difference in controls (p = 0.055) (Fig. 1). The CIMT was significantly thicker in the CHD patients than in the controls (Table 1). The p66shc mRNA and protein expression levels in the PBMs were significantly higher in the CHD group compared with the control group (Fig. 2, 3). The p66shc mRNA expression levels in the CAD and ACS patients were significantly higher than in the controls (1.32 ± 0.76 and 1.44 ± 0.72, respectively, vs. 0.94 ± 0.51; both p \ 0.01); however, there was no significant difference between the CAD and ACS subgroups (p = 0.62). When all of the diabetes mellitus patients in both groups were

Fig. 1 Flow-mediated dilation (FMD) and nitroglycerine-induced dilation (NID) were lower in the CHD patients than in the controls (*p \ 0.001, **p \ 0.05). There is significant difference between FMD and NID in CHD patients (***p \ 0.05)

Fig. 2 The expression of p66shc mRNA relative to the 18S rRNA in the peripheral blood monocytes from CHD patients (n = 78) and controls (n = 38) by real-time RT-PCR

Heart Vessels

Fig. 3 a p66shc Protein expression in the peripheral blood monocytes from CHD patients and controls by Western blot. GAPDH was used as a loading control. b Densitometry by blots (12 CHD patients and 12 controls), as in a Table 2 Univariate analysis in all study subjects for the determinants of the expression of p66shc mRNA in PBMs with some parameters r

p

Age

-0.247

0.124

Smoking

-0.133

0.415

0.160

0.501

Diseased vessels Sex

0.144

0.376

BMI

-0.041

0.800

TG

-0.122

0.454

TC

0.039

0.811

LDL-C

0.886

\0.001

HDL-C Glucose

-0.207 -0.156

0.200 0.335

Hs-CRP

0.155

0.340

Hcy

0.693

\0.001

FMD

-0.910

\0.001

NID

-0.825

\0.001

0.627

\0.001

CIMT

Abbreviations are as shown in Table 1

excluded, the p66shc mRNA expression remained significantly higher in the CHD patients than in the controls (1.27 ± 0.53 vs. 0.95 ± 0.54, p = 0.024). The univariate analysis in all study subjects revealed that the expression of p66shc mRNA in the PBMs was significantly positively correlated with the serum LDL-C and Hcy levels and the CIMT and inversely correlated with the FMD and NID (Table 2). After multiple linear regression analysis, the LDL-C and Hcy levels and the FMD remained independently correlated with p66shc mRNA expression in the PBMs (Table 3).

Discussion In this study, we found that the expression of p66shc mRNA and protein in the PBMs in the CHD patients was significantly higher than in the control subjects. The expression of p66shc mRNA was independently correlated

with the endothelial-dependent function and the LDL-C and Hcy levels. The peripheral monocytes can readily attach to the vascular endothelium when factors that contribute to the progression of atherosclerosis are presented. Thus, monocytes/macrophages play a pivotal role in the pathogenesis of human atherosclerosis [16]. Genetic deletion of p66shc can decrease the superoxide production in macrophages [6] and reduce the content of the macrophage-derived foam cells [8], demonstrating that the function of monocyte/ macrophage was modulated by p66shc expression. Further more, it is easy to collect monocytes from peripheral blood. These are the reasons why we chose to investigate the p66shc expression in PBMs in this study. A number of animal and cell experiments have confirmed that the expression of p66shc is related to vascular atherosclerosis and endothelial dysfunction. Studies that have investigated p66shc mRNA expression in humans are limited. Pagnin et al. [12] first reported that the amount of p66shc mRNA was significantly higher in patients with diabetes mellitus, one of the most common oxidative stress-related diseases, than in healthy subjects. Noda et al. [13] reported an elevation of p66shc mRNA expression in PBMs in CAD patients. Recently, a study by Franzeck et al. [14] showed elevated levels of p66shc mRNA in the PBMs of patients who suffered from an ACS. They found no elevated levels of p66shc mRNA in the PBMs of the patients who displayed stable CAD and in the non-CAD controls, and they hypothesized that the elevated p66shc expression of the CAD patients in the Noda et al. [13] study was associated with the double diabetes patients in the CAD group rather than the presence of stable CAD. Our study found elevated p66shc mRNA in the CAD and ACS patients; however, there was no difference in the elevation between the CAD and the ACS patients. This variance might be explained by the limitations of our study, including a relatively small group of CAD patients, which could prevent the result from reaching statistical significance. We first found that the p66shc protein expression was higher in CHD patients than in the controls by Western

123

Heart Vessels Table 3 Multivariate analysis of the effects factor on p66shc mRNA expression in PBMs Partial regression coefficient

Standard error

Standardized regression coefficient –

t

p

(Constant)

0.650

0.586

1.109

0.275

LDL-C

0.229

0.069

0.327

3.342

0.002

Hcy

0.062

0.018

0.216

3.433

0.002

FMD

-8.100

1.757

-0.466

-4.610

\0.001

NID

-1.254

1.721

-0.072

-0.729

0.471

CIMT

-0.040

0.241

-0.012

-0.166

0.869

Abbreviations are as shown in Table 1

blot. In our study, we did not exclude diabetes patients; however, there is a lack of difference in the prevalence of diabetes between the two groups, and when all of the diabetics are excluded in both groups, the p66shc expression remains statistically higher in the CHD patients than in the controls, demonstrating that the difference is likely attributable to the presence of CHD. Our study and the previous studies support the hypothesis that p66shc, through its proapoptotic and oxidative stress-modulating properties, might play an essential role in atherosclerosis and CHD. Endothelial dysfunction is an initial and key step of atherosclerosis. We hypothesize that upregulated p66shc expression in CHD patients, by increasing the vascular ROS levels and decreasing the vascular NO production, would contribute to impaired endothelial function in these patients. Using noninvasive ultrasound, we found that endothelium-dependent and -independent functions are impaired in CHD patients compared to controls which we had previously analyzed the reasons [15]. For the first time, we demonstrated that the expression of p66shc in PBMs had a significant negative correlation with endotheliumdependent vasodilatation in this study cohort. This finding illustrated that p66shc gene expression might play a pivotal role in the impaired endothelial function of CHD patients. A previous study found that the p66shc gene expression in the PBMs was significantly increased in proportion to the number of diseased vessels [13]. Our result was not in agreement with this finding and failed to find this association. The CIMT is a reliable, reproducible, noninvasive method of detecting and monitoring the progression of atherosclerosis and reflects to a high degree the atherosclerotic condition of the coronary artery [17]. We measured the CIMT and found a lack of correlation with p66shc mRNA expression in a multiple regression analysis. The discrepant results between the previous study and ours might be caused by the different study populations and excluding the ACS patients in unstable and severe condition from our study. We did not clarify the molecular mechanism for the elevation of the p66shc gene expression levels in the CHD

123

patients and the inverse correlation with the endotheliumdependent vasodilatation. However, previous studies have demonstrated that the increased expression of p66shc might be closely related to epigenetic modulation of the promoter P66shc DNA promoter hypomethylation and histone hyperacetylation can induce the up-regulation of the p66shc gene. Treating cells with the DNA methyltransferase inhibitor (DNMTi) 5-Azacytidine (5-AZA) and the histone deacetylase inhibitor (HDACi) Trichostatin (TSA) could induce re-expression in the cells that did not express p66shc [18]. Recently, LDL-C and Hcy, wellknown independent risk factors for atherosclerotic vascular disease and endothelial dysfunction, have been demonstrated to hypomethylate-specific CpG dinucleotides in the human p66shc promoter and dose dependently stimulate p66shc transcription in human endothelial cells [19, 20]. The knockdown of p66shc inhibits the increase in ROS and the decrease in nitric oxide elicited by LDL-C and Hcy, prevents and mitigates LDL-C- and Hcy-induced up-regulation of the endothelial intercellular adhesion molecule-1 and adhesion of monocytes to the endothelial cells [19, 20]. Our study showed that LDL-C and Hcy were independently correlated with the p66shc mRNA levels in the PBMs, and the significant correlation between LDL-C and the p66shc expression is in agreement with a previous study [13]. These findings may in part explain the elevated p66shc expression in the PBMs and the endothelial dysfunction in CHD patients and may indicate that the disordered metabolism of LDL-C and Hcy is likely to regulate p66shc expression. There is an unavoidable limitation in our study that a part of the CHD and control subjects were given statins and angiotensin-converting enzyme inhibitors (ACEI), which both could improve endothelium-dependent vasodilatation. These may contribute to the relatively higher FMD levels in our study and narrow the difference between FMD and NID. In addition, although the taking ratio was similar between the two groups, stain use will still affect the correlation results of LDL-C with the expression of p66shc in both univariate and multiple linear regression analyses and may weaken the role of LDL-C to a certain extent.

Heart Vessels

There are many factors in the development of atherosclerosis and endothelial dysfunction. The expression of p66shc may be a new biomarker predicting the presence of CHD and endothelial dysfunction. A further longitudinal study with a relatively large number of patients will endeavor to elucidate this issue and address whether reduction of p66shc gene expression could prevent endothelial dysfunction-related diseases. Acknowledgments This work was supported by grants from Science and Technology Department of Hunan Province (2012SK3215) and Research Project of Changsha City (K1303025-31). Conflict of interest

None.

References 1. Madamanchi NR, Vendrov A, Runge MS (2005) Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol 25:29–38 2. Yu BP (1994) Cellular defenses against damage from reactive oxygen species. Physiol Rev 74:139–162 3. Landmesser U, Hornig B, Drexler H (2004) Endothelial function: a critical determinant in atherosclerosis? Circulation 109: II27– II33. 4. Okumura K, Imamura A, Murakami R, Numaguchi Y, Matsui H, Toyoaki M (2005) Endothelial function and early atherosclerotic changes. Future Cardiol 1:501–508 5. Migliaccio E, Giorgio M, Mele S, Pelicci G, Reboldi P, Pandolfi PP, Lanfrancone L, Pelicci PG (1999) The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature 402:309–313 6. Tomilov AA, Bicocca V, Schoenfeld RA, Giorgio M, Migliaccio E, Ramsey JJ, Hagopian K, Pelicci PG, Cortopassi GA (2010) Decreased superoxide production in macrophages of long-lived p66Shc knock-out mice. J Biol Chem 285:1153–1165 7. Napoli C, Martin-Padura I, de Nigris F, Giorgio M, Mansueto G, Somma P, Condorelli M, Sica G, De Rosa G, Pelicci P (2003) 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 USA 100:2112–2116 8. Martin-Padura I, de Nigris F, Migliaccio E, Mansueto G, Minardi S, Rienzo M, Lerman LO, Stendardo M, Giorgio M, De Rosa G, Pelicci PG, Napoli C (2008) p66Shc deletion confers vascular protection in advanced atherosclerosis in hypercholesterolemic apolipoprotein E knockout mice. Endothelium 15:276–287 9. Yamamori T, White AR, Mattagajasingh I, Khanday FA, Haile A, Qi B, Jeon BH, Bugayenko A, Kasuno K, Berkowitz DE, Irani K (2005) P66shc regulates endothelial NO production and

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

endothelium-dependent vasorelaxation: implications for ageassociated vascular dysfunction. J Mol Cell Cardiol 39:992–995 Camici GG, Cosentino F, Tanner FC, Luscher TF (2008) The role of p66Shc deletion in age-associated arterial dysfunction and disease states. J Appl Physiol 105:1628–1631 Camici GG, Schiavoni M, Francia P, Bachschmid M, MartinPadura I, Hersberger M, Tanner FC, Pelicci P, Volpe M, Anversa P, Lu¨scher TF, Cosentino F (2007) Genetic deletion of p66(Shc) adaptor protein prevents hyperglycemia-induced endothelial dysfunction and oxidative stress. Proc Natl Acad Sci USA 104:5217–5222 Pagnin E, Fadini G, de Toni R, Tiengo A, Calo` L, Avogaro A (2005) Diabetes induces p66shc gene expression in human peripheral blood mononuclear cells: relationship to oxidative stress. J Clin Endocrinol Metab 90:1130–1136 Noda Y, Yamagishi S, Matsui T, Ueda S, Ueda S, Jinnouchi Y, Hirai Y, Imaizumi T (2010) The p66shc gene expression in peripheral blood monocytes is increased in patients with coronary artery disease. Clin Cardiol 33:548–552 Franzeck FC, Hof D, Spescha RD, Hasun M, Akhmedov A, Steffel J, Shi Y, Cosentino F, Tanner FC, von Eckardstein A, Maier W, Lu¨scher TF, Wyss CA, Camici GG (2012) Expression of the aging gene p66Shc is increased in peripheral blood monocytes of patients with acute coronary syndrome but not with stable coronary artery disease. Atherosclerosis 220:282–286 Zhang X, Zhao SP, Li XP, Gao M, Zhou QC (2000) Endothelium-dependent and -independent functions are impaired in patients with coronary heart disease. Atherosclerosis 149:19–24 Tani S, Matsumoto M, Anazawa T, Kawamata H, Furuya S, Takahashi H, Iida K, Washio T, Kumabe N, Kobori M, Nagao K, Hirayama A (2012) Development of model for prediction of coronary atherosclerotic regression: evaluation of high-density lipoprotein cholesterol level and peripheral blood monocyte count. Heart Vessels 27:143–150 Magda SL, Ciobanu AO, Florescu M, Vinereanu D (2013) Comparative reproducibility of the noninvasive ultrasound methods for the assessment of vascular function. Heart Vessels 28:143–150 Ventura A, Luzi L, Pacini S, Baldari CT, Pelicci PG (2002) The p66Shc longevity gene is silenced through epigenetic modifications of an alternative promoter. J Biol Chem 277:22370–22376 Kim CS, Kim YR, Naqvi A, Kumar S, Hoffman TA, Jung SB, Kumar A, Jeon BH, McNamara DM, Irani K (2011) Homocysteine promotes human endothelial cell dysfunction via site-specific epigenetic regulation of p66shc. Cardiovasc Res 92:466–475 Kim YR, Kim CS, Naqvi A, Kumar A, Kumar S, Hoffman TA, Irani K (2012) Epigenetic up-regulation of p66shc mediates low density lipoprotein (LDL) cholesterol-induced endothelial cell dysfunction. Am J Physiol Heart Circ Physiol 303:H189–H196

123