Published online September 1, 2008
PEDIATRICS Vol. 122 No. 3 September 2008, pp. e648-e655 (doi:10.1542/peds.2008-0735)
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ARTICLE

Oxidative Stress Is Associated With Arterial Dysfunction and Enhanced Intima-Media Thickness in Children With Hypercholesterolemia: The Potential Role of Nicotinamide-Adenine Dinucleotide Phosphate Oxidase

Francesco Martino, MDa, Lorenzo Loffredo, MDb, Roberto Carnevale, PhDb, Valerio Sanguigni, MDc, Eliana Martino, MDa, Elisa Catasca, MSa, Cristina Zanoni, MSa, Pasquale Pignatelli, MDb and Francesco Violi, MDb

a Center of Clinic Lipid Research, Department of Pediatrics
b Division of Internal Medicine H, Department of Experimental Medicine, University of Rome "La Sapienza," Rome, Italy
c Department of Internal Medicine, University of Rome "Tor Vergata," Rome, Italy


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
BACKGROUND. Endothelial dysfunction and intima-media thickness are precocious manifestations of hypercholesterolemia, but the mechanism is unclear.

OBJECTIVE. The aim of the study was to analyze the interplay among endothelial dysfunction, intima-media thickness, and oxidative stress in children with hypercholesterolemia.

METHODS. We performed a cross-sectional study comparing flow-mediated dilation, intima-media thickness, lipid profile, urinary isoprostanes as markers of oxidative stress, and platelet expression of gp91phox, the catalytic unit of nicotinamide-adenine dinucleotide phosphate oxidase, in a population of 50 children with hypercholesterolemia (mean age ± SD: 10.0 ± 3.7 years) and 50 children without hypercholesterolemia (mean age: 9.2 ± 3.5 years). Four children with hereditary deficiency of gp91phox were studied also.

RESULTS. Children with hypercholesterolemia had reduced flow-mediated dilation (mean ± SD: 6.2 ± 2.4 vs 9.2 ± 2.5%) and enhanced intima-media thickness (0.45 ± 0.07 vs 0.40 ± 0.06 mm), urinary isoprostanes (86.9 ± 51.6 vs 45.9 ± 25.6 pg/mg creatinine), and gp91phox platelet expression (4.4 ± 3.8 vs 2.0 ± 1.7 mean fluorescence) compared with control subjects. At bivariate analysis, flow-mediated dilation was correlated with low-density lipoprotein cholesterol, intima-media thickness, urinary isoprostanes, and platelet gp91phox. Stepwise multiple linear regression analysis showed that, in children with hypercholesterolemia, flow-mediated dilation and intima-media thickness were significantly associated with low-density lipoprotein cholesterol and urinary isoprostanes; also, gp91phox platelet expression was an independent predictor of urinary isoprostanes. Children with gp91phox hereditary deficiency showed downregulation of platelet gp91phox and reduced urinary excretion of isoprostanes.

CONCLUSIONS. The study suggests that gp91phox-mediated oxidative stress may have a pathogenic role in the anatomic and functional changes of the arterial wall occurring in children with premature atherosclerosis.

Key Words: atherosclerosis • hypercholesterolemia • oxidative stress • flow-mediated dilation • gp91phox

Abbreviations: TC—total cholesterol • LDL—low-density lipoprotein • IMT—intima-media thickness • HC—children with hypercholesterolemia • FMD—flow-mediated dilation • NO—nitric oxide • ROS—reactive oxygen species • NADPH—nicotinamide-adenine dinucleotide phosphate • NC—children without hypercholesterolemia • IgG—immunoglobulin G • FITC—fluorescein isothiocyanate • X-CGD—X-linked chronic granulomatous disease

Atherosclerosis is a process that is already detectable in children with classic risk factors for atherosclerosis. Autopsy studies performed in children or youth with established risk factors demonstrated a positive association with the presence and extent of atherosclerotic lesions in the aorta and coronary arteries.1–3 Among the classic risk factors for atherosclerosis, total cholesterol (TC) seems to be a major determinant of early atherosclerosis. Thus, autopsy studies documented an association between TC and low-density lipoprotein (LDL) cholesterol with the extent and severity of atherosclerosis in infants, children, and adolescents.1–3 An early increase of cholesterol is also relevant in the progression of atherosclerosis in adults; thus, measurement of LDL in childhood predicts carotid intima-media thickness (IMT) in young adults.4

In children with hypercholesterolemia (HC), early functional and anatomic changes of the arterial wall have been detected.5–14 In particular, a significant decrease of flow-mediated dilatation (FMD) has been observed, suggesting a role for cholesterol in impairing the nitric oxide (NO) release or inhibiting endothelium NO synthase.5–8 However, the underlying mechanism is unclear. Reactive oxygen species (ROS) seem to be implicated, because they can inhibit NO release or NO synthase.15 In the artery from hypercholesterolemic animals, there is an enhanced production of superoxide anion,15 a reactive oxidant species that rapidly inactivates NO and inhibits NO synthase.15 In accordance with these data, patients with hypercholesterolemia have enhanced oxidative stress and low FMD, suggesting an interplay between these changes.16 Consistent with this suggestion is the demonstration that, in patients with or at risk of atherosclerosis, oxidative stress negatively correlated with FMD and antioxidant treatment was able to restore arterial dilatation.17

In the early phase of atherosclerosis, systemic signs of enhanced oxidative stress have been recently detected,18 but it has never been investigated whether an interplay between enhanced ROS formation and changes in FMD does exist in children with hypercholesterolemia. It also unclear whether oxidative stress has some role in the early anatomic changes, such as increased IMT, occurring in patients with premature atherosclerosis.19 Therefore, we undertook a clinical study in which oxidative stress, FMD, and IMT have been investigated in a population consisting of children with and without high cholesterol levels. To investigate the mechanism potentially accounting for enhanced oxidative stress, we measured the cellular expression of gp91phox, the catalytic core of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, which represents one of the most important cellular sources of superoxide anion.20 These experiments have been conducted by measuring gp91phox on the surface of platelets that have been shown to express several subunits of NADPH oxidase, including its catalytic core.21


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Subjects and Family Classification
A total of 150 subjects, including 50 HC and 50 children without hypercholesterolemia (NC), were recruited through a screening program of dyslipidemia in childhood. They were referred to the lipid research clinic at the University "La Sapienza" for the presence in the children (aged 2–18 years) of abnormal lipid values detected during an occasional laboratory test. The phenotype of the hypercholesterolemic children was classified as follows: family hypercholesterolemia (n = 24) on the basis of the presence of a first-degree relative with hypercholesterolemia (TC >90th age- and gender-specific percentile); family combined hyperlipidemia (n = 6) defined as a presence of a first-degree relative with high triglycerides and/or high TC (TC >90th age- and gender-specific percentile); and polygenic hypercholesterolemia on the basis of the presence of TC >90th age- and gender-specific percentile and without clear family transmission (n = 20).22 Exclusion criteria for all subjects included subjects <2 and >18 years old and the existence of hypothyroidism, renal disease, malignancy, treatment with immunosuppressive drugs, connective tissue disease, and acute illness.

Both patients and control subjects were recruited from the same geographic area and followed a typical Mediterranean diet. None of the patients had clinical evidence of cardiovascular disease (as shown by clinical history, physical examination, or ECG), diabetes mellitus, or hypertension. Patients with hypercholesterolemia had not taken any lipid-lowering agents or antiplatelet drugs in the previous 30 days.

Blood samples were obtained from an antecubital vein after an overnight fast. Serum TC, high-density lipoprotein cholesterol, and triglycerides were measured by an Olympus AN 560 apparatus (Olympus, Olympus Optical Co, Suzuka, Japan) using an enzymatic colorimetric method. LDL cholesterol levels were calculated according to the Friedewald formula. The hospital ethics committee approved the study protocol, and written informed consent was obtained from all of the patients.

Measurement of Carotid IMT and FMD
Ultrasound assessment of IMT and FMD was investigated as described previously.23

Urinary 8-iso-prostaglandin F2{alpha} Assays
Urinary 8-iso-prostaglandin F2{alpha} was measured by the previously described and validated enzyme immunoassay assay method.24,25 Ten-mL urine aliquots were extracted on a C-18 SPE column; the purification was tested for recovery by adding a radioactive tracer (tritiated 8-iso-prostaglandin F2{alpha}, Cayman Chemical, Ann Arbor, MI). The eluates were dried under nitrogen, recovered with 1 mL of buffer, and assayed in a 8-iso-prostaglandin F2{alpha}-specific enzyme immunoassay kit (Cayman Chemical). 8-Iso-prostaglandin F2{alpha} concentration was corrected for recovery and creatinine excretion and expressed as picograms per milligram of creatinine. Intra-assay and interassay coefficients of variation were 2.1% and 4.5%, respectively.

Platelet gp91phox Expression
Sample preparation was started immediately after blood sampling without any stirring, centrifugation, or washing steps to prevent artificial platelet activation. The citrate anticoagulated whole blood (9 parts blood and 1 part 3.8% Na citrate) was diluted with Tyrode's buffer containing 0.2% bovine serum albumin, 5 µM glucose, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.35). A working solution of ThromboFix Platelet Stabilizer26 was prepared according to the manufacturer's instructions. Fixation of the samples was initiated by adding 250 µL of the blood to an equivalent volume of the platelet stabilizer for 30 minutes at room temperature. Platelet gp91phox was expressed as mean fluorescence.

Antibody Labeling
To measure the gp91phox expression on platelets, a blood sample volume of 50 µL, fixed previously, was incubated with 10 µL (2 µg/mL) of unconjugated antibody anti-gp91phox. After a 30-minute incubation at room temperature, the sample was incubated with the respective secondary antibody donkey anti-goat immunoglobulin G (IgG) fluorescein isothiocyanate (FITC) for 30 minutes. For the detection of platelets, 10 µL of monoclonal antibody CD61 phycoerythrin (2 µg/mL) were added to the same sample.

The anti-gp91phox and anti-CD61 phycoerythrin antibodies were replaced by nonspecific donkey anti-goat IgG FITC and IgG1 phycoerythrin, respectively, with the same ratio for FITC and phycoerythrin for isotype controls. Adequate concentration of the polyclonal antibody had been defined by preliminary testing. After incubation, samples were diluted with 1 mL of phosphate-buffered saline buffer and analyzed with flow cytometry.

Flow Cytometry
All of the samples were analyzed within 15 minutes after the final dilution on an Epics XL-MCL cytometer (Coulter Electronics) equipped with an argon laser at 488 nm. FITC was detected at 825 to 850 nm, phycoerythrin at 875 to 900 nm. All of the parameters were collected with a 4-decade logarithmic amplification. Spectral overlap was balanced by fluorescence compensation, as defined in preliminary tests. Cytometer settings were checked with Flow-Chek Fluorospheres. Platelets and platelet-platelet aggregates were identified by logical gating according to their CD61 phycoerythrin fluorescence and forward scatter characteristics. A threshold of ~0.5% FITC-positive events was set in the first isotype control of each subject. Analysis was stopped automatically after the measurement of 50 000 events. Intra-assay and interassay coefficients of variation were 1.0% and 0.2%, respectively.

Patients With X-Linked Chronic Granulomatous Disease
X-linked chronic granulomatous disease (X-CGD) is a rare (prevalence 1:1 000 000 individuals) primary immunodeficiency affecting the innate immunologic system; X-CGD is characterized by life-threatening bacterial and fungal infections.27 It is caused by mutations in any of the 4 genes encoding subunits of the O2– generating NADPH oxidase, resulting in defective O2– generation and intracellular killing.28 We identified 4 male patients (mean age ± SD: 12.2 ± 2.5 years) with hereditary deficiency of gp91phox that was diagnosed as described previously.29

Statistical Analysis
Comparisons between groups were conducted by using Student's t test. Data are presented as means ± SDs. Proportions and categorical variables were tested by using the {chi}2 test. The correlation analysis was conducted by using Pearson's test. Multiple linear regression analysis was performed by using a stepwise selection method and was performed to determine the independent parameters of FMD, IMT, and isoprostanes. Statistical significance was defined as P < .05. Statistical analysis was performed with SPSS 13.0 for Windows (SPSS Inc, Chicago, IL).

We computed the minimum sample size with respect to a 2-tailed 1-sample Student's t test, considering: (1) a clinically relevant difference for FMD variation to be detected between the HC and NC {delta} ≥ 2.0%; (2) SDs were homogeneous between groups with SDs equal to 2.5%; and (3) a type I error probability {alpha} value at .05 and power 1 – β value at .90. This resulted in a minimum sample size of 34 for the group.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Clinical characteristics of HC and NC are reported in Table 1. No significant difference was found between the 2 groups for systolic and diastolic blood pressure, fasting blood glucose, age, and BMI (Table 1).


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  		TABLE 1 Clinical Characteristics of HC and NC

 
Endothelial Dysfunction in HC
Compared with NC, HC had lower FMD (Table 1 and Fig 1A). In HC, FMD was significantly associated with IMT, total and LDL cholesterol, urinary isoprostanes (Table 2 and Fig 2A–C), and platelet gp91phox (Table 2). Conversely, in NC, FMD was associated only with IMT (Table 2 and Fig 2A–C).


Figure 1
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  		FIGURE 1 FMD (A), IMT (B), isoprostanes (C), and gp91phox (D) values in HC and NC. aP ≤ .005.

 

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  		TABLE 2 Simple Linear Regression Analysis for Determinants of FMD, IMT, and Urinary Isoprostanes in HC and NC

 

Figure 2
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  		FIGURE 2 Simple linear regression analysis between FMD and IMT (A), FMD and LDL (B), FMD and isoprostanes (C), IMT and LDL (D), IMT and isoprostanes (E), and isoprostanes and gp91phox (F) in HC and NC.

 
To establish the independent predictors of FMD in HC, a multiple linear regression analysis, including the variables linearly associated with the dependent variable (Table 2), was performed; because TC and LDL cholesterol were highly correlated (r = 0.930; variance inflation factor: 7.432), generating a multicollinearity problem, we included only LDL cholesterol as an independent variable in the regression model. The independent predictive variables associated with FMD were LDL cholesterol (SE = .007; standardized coefficient β = –.424; P = .001) and urinary isoprostanes (SE = .006; standardized coefficient β = –.394; P = .002; R2 = 60%).

IMT in HC
Compared with NC, HC had a higher IMT (Table 1 and Fig 1B). In HC, bivariate analysis showed that IMT was significantly associated with FMD, total and LDL cholesterol, and urinary isoprostanes (Table 2 and Fig 2A, D, and E). Also in NC, IMT was associated with FMD and urinary isoprostanes (Table 2 and Fig 2A, D, and E).

To establish the independent predictors of IMT in HC, a multiple linear regression analysis, including the variables linearly associated with the dependent variable (Table 2), was performed; for the reason outlined above, we included only LDL cholesterol as an independent variable in the regression model. The independent predictive variables associated with IMT were LDL cholesterol (SE = .001; standardized coefficient β = .308; P = .016) and urinary isoprostanes (SE = .001; standardized coefficient β = .412; P = .002; R2 = 29%).

Oxidative Stress in Children With High and Normal Cholesterol and Patients With X-CGD
Compared with NC, HC had higher values of urinary isoprostanes and gp91phox platelet expression (Table 1 and Fig 1C and D). In HC, isoprostanes were significantly associated with IMT, FMD, and gp91phox (Table 2 and Fig 2C, E, and F). In NC, urinary isoprostanes were associated with IMT and gp91phox (Table 2 and Fig 2C, E, and F).

To establish the independent predictors of urinary isoprostanes in HC, a multiple linear regression analysis including the variables linearly associated with the dependent variable (Table 2) was performed; the independent predictive variables associated with urinary isoprostanes were platelet gp91phox (SE = 1.164; standardized coefficient β = .539; P < .001) and FMD (SE = 2.477; standardized coefficient β: –.218; P = .01; R2 = 43%).

Patients With X-CGD
Four children with hereditary deficiency of gp91phox (mean age: 12.25 ± 2.5 years) were investigated to validate the methodology used to measure the platelet expression of gp91phox. In these children, platelet expression of gp91phox (0.95 ± 0.27 mean fluorescence) was downregulated compared with HC and NC; also, the urinary excretion of isoprostanes (6.92 ± 3.44 pg/mg creatinine) was less compared with that of the other 2 groups. Furthermore, patients with X-CGD had a higher FMD compared with NC (13.0% ± 5.9% vs 9.2% ± 2.5%, P = .012) and HC (13.0% ± 5.9% vs 6.2% ± 2.4%; P < .001). Patients with X-CGD had lower IMT compared with NC and HC, but the difference was not significant (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
The study provides the first evidence of an early relationship between oxidative stress and anatomic and functional changes of the arterial wall in HC and suggests that activation of gp91phox plays a role in enhancing oxidative stress. Previous studies have shown a reduced FMD not only in adults but also in HC, thereby suggesting that cholesterol elicits early functional changes of the arterial wall.5–8 FMD is prevalently dependent on NO release from endothelium, as also suggested by the significant correlation between FMD and serum plasma nitroso compounds.30 Oxidative stress seems to play a pivotal role in modulating FMD, likely via interfering with NO bioavailability and biosynthesis.31 Thus, oxidative stress and FMD are inversely related in patients with or without atherosclerosis, and acute infusion of an antioxidant such as vitamin C is associated with rapid restoration of arterial function.17 Children with hypercholesterolemia have already been shown to have enhanced oxidative stress and impaired FMD, but the interplay between them is still unclear. The present study demonstrated that HC have enhanced urinary excretion of isoprostanes, a finding that reinforces our previous data showing an early increase of oxidative stress in this setting.18 To the best of our knowledge, there has been only 1 study that explored the behavior of isoprostanes in HC32; at variance with our data, the authors did not find any changes in urinary isoprostanes compared with control subjects, but it cannot be excluded that inadequate storage of urinary samples (–20°C instead of –80°C) could have precluded reliable analysis.

The inverse correlation between urinary isoprostanes and FMD lends support to the hypothesis that oxidative stress may be implicated in the early dysfunction observed in children with hypercholesterolemia. Our finding is apparently at variance with a previous study showing no relationship between FMD and oxidative LDL, another marker of oxidative stress in children at risk of atherosclerosis33; it is unclear whether this may depend on the different methodology used to measure oxidative stress or on the small sample size of the study that included only 14 patients with familiar hypercholesterolemia. Conversely, our results provide support for a previous study showing that the combination of 2 antioxidants, namely, vitamins E and C, was associated with improvement of FMD in HC.34 It should be noted, however, that, in this study, no changes of isoprostanes were observed after antioxidant vitamins.34 However, isoprostanes were measured in serum but not in urine, which is a much more reliable assay.34

In accordance with previous studies,9–14 we found enhanced IMT in HC compared with control subjects, indicating that anatomic and functional changes of the arterial wall coexist in the early phase of atherosclerosis. A novel finding of the study was that urinary isoprostanes are determinant of IMT, suggesting a role for oxidative stress not only in impairing FMD but also in provoking early anatomic changes of the arterial wall. This finding is consistent with previous studies in adults showing a direct association between oxidative stress and IMT.35–37

In this study we also investigated the mechanism accounting for enhanced oxidative stress in HC. NADPH oxidase is an important cellular source of ROS with a potential pathophysiologic role in the atherosclerotic process.20 Several subunits of the enzyme, including gp47phox, gp22phox, and gp91phox are overexpressed in animal and human atherosclerosis, where they likely contribute to the increased arterial production of superoxide anion.38 We have hypothesized recently that this enzyme may have an important role in systemic oxidative stress, because adult patients with hereditary deficiency of gp91phox (X-CGD) have reduced urinary excretion of isoprostanes and no increase of oxidative stress in a clinical model of ischemia-reperfusion.39 These data prompted us to speculate that NADPH oxidase may be relevant for the enhanced oxidative stress in HC and, hence, could be responsible for early increased isoprostane formation. We used platelets as a tool to investigate this issue for several reasons. First, platelets express gp91phox, which plays an important role in superoxide anion production as documented by the fact that, in patients with hereditary deficiency of gp91phox, the platelet production of this oxygen radical is almost absent.29 Second, on stimulation, platelets produce isoprostanes40 and can, therefore, contribute to the isoprostane circulating levels. Third, platelets represent a relatively simple noninvasive tool to measure the regulation of NADPH oxidase in clinical settings at risk or with atherosclerosis.

On the basis of these arguments, we developed a method to measure gp91phox on the platelet surface in patients with potentially different gp91phox regulation, and we observed that the assay was sensitive to quantify the expression of gp91phox on the platelet surface. Even if the analysis was performed in only 4 children with hereditary deficiency of gp91phox, it is noteworthy that, in these children, platelet expression of gp91phox was downregulated compared with healthy subjects; conversely, platelet expression of gp91phox was upregulated in HC. This finding indicated that the upregulation of NADPH oxidase is an early phenomenon potentially contributing to the early phase of atherosclerosis via enhancing oxidative stress. Consistent with such hypothesis was the demonstration that platelet gp91phox was a major determinant of urinary isoprostanes; urinary isoprostanes were very low in children with hereditary deficiency of NADPH oxidase and gradually increased from NC to HC, suggesting that the enhanced oxidative stress observed in hypercholesterolemia may be mediated by NADPH oxidase upregulation.

The study has some implications and limitations that should be acknowledged. Despite the fact that platelets produce isoprostanes40 and express platelet gp91phox, the contribution of other cells, including phagocytes and endothelial cells, in enhancing oxidative stress should be investigated. In this context, it would be of interest to analyze whether platelet gp91phox mirrors the expression of this NADPH oxidase subunit on endothelial cells.

The direct correlation between platelet gp91phox and urinary isoprostanes, along with the low urinary excretion of isoprostanes in patient knockout for this NADPH oxidase subunit, reinforces our hypothesis39 that NADPH oxidase could be a major determinant of isoprostane formation. Additional study in a larger X-CGD population should be performed to provide definite support to such hypothesis.

The mechanism accounting for NADPH oxidase activation was not investigated in the present study. Arachidonic acid that is increased in the platelets of hypercholesterolemic patients41,42 plays an important role in NADPH oxidase activation.43 Additional study is necessary to see if in hypercholesterolemia NADPH oxidase activation is arachidonic acid mediated.


   CONCLUSIONS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
This study suggests that, in HC, oxidative stress represents a common pathway eliciting endothelial dysfunction and enhanced IMT. Upregulation of NADPH oxidase expression seems to be implicated in early oxidative stress and could provide a novel insight in the mechanism accounting for premature atherosclerosis.


   ACKNOWLEDGMENTS
 
This study was supported in part by a grant from the University of Rome "La Sapienza" (Ateneo 2006) to Dr Violi.

We are grateful to Plebani Alessandro and Soresina Anna Rosa for recruitment of patients with X-CGD and to Ludovica Perri, Elisa Frusone, and Simona Bartimoccia for technical assistance and fruitful collaboration.


   FOOTNOTES
 
Accepted May 14, 2008.

Address correspondence to Francesco Violi, MD, IV Divisione di Clinica Medica, Viale del Policlinico 155, Rome, 00161, Italy. E-mail: francesco.violi{at}uniroma1.it

The authors have indicated they have no financial relationships relevant to this article to disclose.

Drs Martino and Loffredo contributed equally to this work.


What's Known on This Subject

Endothelial dysfunction and IMT are precocious manifestations of hypercholesterolemia, but the mechanism is unclear.

 

What This Study Adds

The study suggests that gp91phox-mediated oxidative stress may have a pathogenic role in the anatomic and functional changes of the arterial wall occurring in children with premature atherosclerosis.

 


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 

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PEDIATRICS (ISSN 1098-4275). ©2008 by the American Academy of Pediatrics




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