Oxidative stress in carotid arteries of patients submitted to carotid endarterectomy . The role of aging process

PURPOSE: To evaluated the role of oxidative stress on aging process in patients submitted to carotid endarterectomy. METHODS: Twenty patients were divided into two groups: older group (≥ 70 years old); and the younger group (< 70 years old). We evaluated the reactive oxygen species (ROS) concentration, nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, superoxide dismutase (SOD) and catalase (CAT) activities as so as nitrite levels in fragments of carotid arteries harvested during carotid endarterectomy for treatment of high grade carotid stenosis. RESULTS: We observed a higher levels of ROS and NADPH oxidase activity in the older group (p<0.05). Furthermore, the nitrite concentration was lower in the older group (14.55 ± 5.61 x 10-3 versus 26.42 ± 8.14 x 10-3 μM; p=0.0123). However, the activities of antioxidant enzymes (CAT and SOD) were similar in both the groups. CONCLUSIONS: Arterial aging is associated with increased concentrations of oxygen species and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity as so as nitrite reduction in human carotid artery specimens. Maybe therapies that block NADPH oxidase activity and enhance nitrite stores would be a good strategy to reduce the effect of oxidative stress in arterial aging.


Introduction
Atherosclerotic disease of the carotid arteries is responsible for a considerable number of all strokes and increases with aging 1,2 .Oxidative stress, defined as an imbalance between oxidants and antioxidants in favor of the former, plays a significant role in the pathogenesis of atherosclerotic vascular disease as well as an important determinant in the aging process 3 .Reactive oxygen species (ROS) such as superoxide (•O 2 -) and hydrogen peroxide (H 2 O 2 ) contribute to the oxidative stress damage and can be scavenged by superoxide dismutase (SOD) and catalase (CAT), respectively 4,5 .In the experimental setting, there is some evidence that enzyme responsible for •O 2 -production in the atherosclerotic arteries is the NADPH oxidase 6 .This enzyme is membrane bound and generates superoxide by transferring electrons from NADPH to molecular oxygen 7 .Furthermore, nitric oxide (NO), also known as endothelium-derived relaxation factor, is produced in various types of cells and it inhibits platelet aggregation, induces vascular relaxation, and impairs vascular smooth muscle cell proliferation 8,9 .NO is thought to exert vasculoprotective and antiapoptotic effects and decreased of its bioavailability has been linked to cardiovascular aging 10 .Superoxide anion reacts with NO causing peroxynitrite (•ONOO -) generation, which is a well known radical which causes endothelial dysfunction 10 .
There are few studies assessing the oxidative stress in human carotid arteries 8,11 .Thereby, we investigated the role of aging on oxidative stress in human carotid arteries of patients submitted to carotid endarterectomy.

Methods
The study was approved by the local Ethics Committee and all participants gave written informed consent.
Ten patients with 70 years or older ("older group") were compared to 10 patients under 70 years old ("younger group").
All the patients were submitted to carotid endarterectomy to treat a high degree carotid stenosis provoked by atherosclerosis.The procedures were performed under general anesthesia and fragments of carotid arteries were prospectively collected to analyze the oxidative stress parameters described below.Demographic data (age and sex) and presence of symptoms were recorded.Computed tomography (CT) angiography of carotid arteries was performed in all patients.Degree of stenosis was determined according to North American Symptomatic Carotid Endarterectomy Trial (NASCET) criteria 12 .

Tissue collection and preparation
The human carotid specimens were removed in the operating room and were immediately stored at -70°C for further analysis of oxidative stress parameters.Carotid samples were macerate in liquid nitrogen.After that, it was homogenized (KCl 150mmol/L; phenyl-methyl-fluoro-sulfonyl 20mmol/L, 1:100) in the Ultra-Turrax homogenizer.Posteriorly, it was performed a sonification with the Hielscher Ultrasound Technology device 13 .

Measurement of oxidative stress parameters
Reactive oxygen species (ROS) concentration was measured by DCFH-DA fluorescence emission (Sigma-Aldrich, USA).Dichlorofluorescein diacetate is membrane permeable and is rapidly oxidized to the highly fluorescent 2,7-dichlorofluorescein (DCF) in the presence of intracellular ROS.The samples were excited at 488 nm and emission was collected with a 525 nm long pass filter.It was expressed as nmols per milligram of protein 14 .Measurement of NADPH oxidase activity was assayed with spectrophotometric method 15 .Nitrites levels were determined using the Griess reagent, in which a chromophore with a strong absorbance at 542 nm is formed by reaction of nitrite with a mixture of naphthyletilenediamine (0.1%) and sulphanilamide (1%).The absorbance was measured in a spectrophotometer to give the nitrite concentration 16 .

Antioxidant enzymes activities
Superoxide dismutase (SOD) activity, expressed as U/mg protein, was based on the inhibition of superoxide radical reaction with pyrogallol 17 .Catalase (CAT) activity was determined by following the decrease in 240nm absorption of hydrogen peroxide (H 2 O 2 ).It was expressed as nmoles/mg protein 18 .Determination of protein concentration was measured by the method of Lowry et al. 19 , using bovine serum albumin as standard.

Statistical analysis
All data are expressed as the mean ± standard error.Comparisons between the groups were analyzed with the Student's t test and exact Fisher's test.A value of p<0.05 was considered significant.

Results
Demography data are summarized in Table 1.Average was significantly higher in the older group.There was no difference in the male/female proportion and presence of symptoms between the groups.Degree of carotid stenosis also were similar between the older and younger groups (78.12 ± 11.7% vs 83.12 ± 11.43%, respectively; p=0.432).Oxidative stress parameters are demonstrated in the Figure 1 (A-E).In the older patients, we observed a higher levels of ROS and an increase in the NADPH oxidase activity in their carotid arteries when compared with young group (p<0.05).
Furthermore, the nitrite concentration was lower in the older group when compared with the younger group (14.55 ± 5.61 x 10 -3 vs 26.42 ± 8.14 x 10 -3 M; p=0.0123).However, the activities of antioxidant enzymes (CAT and SOD) were similar in both the groups.

Discussion
This preliminary study demonstrated that oxidative stress, evaluated by ROS concentration and NADPH oxidase activity were increased in human carotid arteries of older patients (≥70 years old) when compared with younger patients (< 70 years old).It has been demonstrated that arterial oxidative stress develops with aging as a consequence of excessive production of superoxide by NADPH oxidase (20).In the study of Sindler et al. 21, there was about 100% greater of •O 2 -production in the aorta of old mice compared with young control group.Furthermore, Csiszar et al. 10 observed an attenuated production of ROS in mouse aortas of longer-lived mice when compared to shorterlived mice, but expression of NADPH oxidase was similar in both groups.Elevated levels of oxidative DNA damage yet were found in human atherosclerotic plaques 22 .Nevertheless, there are few studies in human beings reporting the role of oxidative stress in the atherosclerosis processes 23,24 .
It has been accepted that ROS are involved in the oxidase in human coronary arteries with evident atherosclerotic plaques, as well as in peripheral arteries and veins without plaques but characterized by systemic endothelial dysfunction 29 .Sindler et al. 21also demonstrated an increase of NADPH oxidase expression in the aorta of old compared with young control mice.These same authors also demonstrated a reduced of SOD activity in aortas of old mice 21 .Superoxide anion can be metabolized in H 2 O 2 by SOD enzyme 10,30 .By converting hydrogen peroxide into water, CAT constitutes a primary antioxidant defense system and could protect cells from ROS and its deleterious consequences 31 .In experimental setting, CAT leads to a delayed onset of atherosclerosis in a murine model 32 .Besides, longer-liver mice have more abundant expression of CAT in aortas when compared to shorter-lived mice, but expression of SOD was similar in both groups 10 .We did not demonstrate any difference in CAT and SOD activities between older and younger patients.In human beings, it has been shown that atherosclerotic plaques had low levels of antioxidant enzymes, but data involving CAT expression are scarces 11 .
The knowledge that ROS are involved in the pathogenesis of vascular disease should stimulate the development of therapies to minimize its effects.Therefore, targeting ROS production by blocking the activity of some enzymes -such as NADPH oxidase -is likely to be a far superior approach for combating oxidative stress and preventing the progression of vascular disease than previous attempts aimed at scavenging ROS 33 .
A rationale for therapies that block NADPH oxidase activity and enhance nitrite stores would be a good strategy to reduce the effect of oxidative stress in arterial aging.We know that this study is bring a preliminary result with a small sample size of patients which could restrict our interpretation of the results, with a potential bias for analysis.We need more robust studies to confirm our findings so that it can be justified any type of antioxidant treatment in patients with carotid disease.

Conclusion
Our results indicate that arterial aging is associated with increased concentrations of oxygen species and nicotinamide adenine dinucleotide phosphate and oxidase activity as so as nitrite reduction in arterial specimens of patients undergoing carotid endarterectomy to high grade stenosis of the carotid arteries.

development of atherosclerosis 25 .
More recently, ROS have been also implicated in other pathological processes in the vessel wall, including endothelial dysfunction, activation of matrix metalloproteinases, and vascular smooth muscle cell (VSMC) migration 26 .Other mechanism by which ROS can promote vascular disease is via reduction of NO bioavailability resulting in lower vasodilator and antiapoptotic action of NO 10,27 .In our study, we observed a significant reduction in nitrite concentration in carotid arteries in the older group compared with young patients.Aging cell nitrite demonstrated that sodium nitrite treatment ameliorated carotid artery endothelial dysfunction in old C57BL6 mice 21 .Moreover, the diminished nitrite concentration in atherosclerotic arteries it is related with enhanced •O 2 -production 8 .In an experimental study, it was observed that nitrite concentrations were lower in old compared with young control animals.These results show that nitrite concentrations in carotid arteries decrease with aging, according to our results 21 .NADPH oxidase is an enzyme with the primary function of generating •O 2 -.Arterial oxidative stress develops with aging as a consequence of excessive production of •O 2 -by NADPH oxidase mainly 20,21 and possibly via selective reductions in antioxidant enzymes, including SOD 21 .In our experiment, we demonstrated an elevated activity of NADPH oxidase in carotid arteries of older patients when compared with youngers.Guzik et al. 28 showed that vascular NADPH oxidases are major sources of •O 2 -in human vessels and showed an association between enzymatic activity and clinical risk factors in atherosclerosis.It has characterized NADPH

TABLE 1 -
Demographic and degree of stenosis of patients submitted to carotid endarterectomy.