Effects of Antioxidants (AREDS Medication) on Ocular Blood Flow and Endothelial Function in an Endotoxin-Induced Model of Oxidative Stress in Humans

Abstract

Purpose.: The Age-Related Eye Disease Study (AREDS) has shown that supplementation of antioxidants slows the progression of age-related macular degeneration (AMD). The mechanism underlying this therapeutic effect may be related to a reduction of reactive oxygen species (ROS). The authors have recently introduced a model showing that the response of retinal blood flow (RBF) to hyperoxia is diminished by administration of lipopolysaccharide (LPS). In the present study, the hypothesis was that this response can be restored by the AREDS medication.

Methods.: Twenty-one healthy volunteers were included in this randomized, double-masked, placebo-controlled, parallel group study. On each study day, RBF and the reactivity of RBF to hyperoxia were investigated before and after infusion of 2 ng/kg LPS. Between the two study days, subjects took either the AREDS medication or placebo for 14 days.

Results.: After administration of LPS reduced retinal arterial vasoconstriction during hyperoxia (AREDS group: 12.5% ± 4.8% pre-LPS vs. 9.4% ± 4.6% post-LPS; placebo group: 9.2% ± 3.3% pre-LPS vs. 7.1% ± 3.5% post-LPS) and a reduced reactivity of RBF during hyperoxia (AREDS: 50.4% ± 8.9% vs. 44.9% ± 11.6%, placebo: 54.2% ± 8.6% vs. 46.0% ± 6.9%) was found. The reduced responses were normalized after 2 weeks of AREDS antioxidants but not after placebo (vasoconstriction: 13.1% ± 4.5% vs. 13.1% ± 5.0% AREDS, 11.2% ± 4.2 vs. 7.4% ± 4.2% placebo; RBF: 52.8% ± 10.5% vs. 52.4% ± 10.5% AREDS, 52.4% ± 9.3% vs. 44.2% ± 6.3% placebo).

Conclusions.: The sustained retinal vascular reaction to hyperoxia after LPS in the AREDS group indicates that antioxidants reduce oxidative stress–induced endothelial dysfunction, possibly by eliminating ROS. The model may be an attractive approach to studying the antioxidative capacity of dietary supplements for the treatment of AMD (ClinicalTrials.gov number, NCT00431691).

Oxidative stress, which refers to cellular damage caused by reactive oxygen species (ROS), has been implicated to contribute to cellular damage in many disease and aging processes. An increased exposure of vascular tissue to ROS is associated with endothelial dysfunction, which is characterized by reduced dilator function, increased inflammatory cell and platelet adhesion, and increased coagulation activity. Evidence from the literature supports the role of oxidative stress in the pathophysiology of several ocular diseases such as diabetic retinopathy, ^1–2 cataract, ³ uveitis, ⁴ retrolental fibroplasia, ⁵ age-related macular degeneration (AMD), ⁶ and possibly glaucoma. ⁷ The retina is especially susceptible to oxidative stress because of its high consumption of oxygen, its high concentration of polyunsaturated fatty acids, and its direct exposure to light. 

Several studies have found a beneficial effect of antioxidants in some of these diseases. Treatment with vitamin C has been reported to reduce retinal vascular leakage in patients with diabetes mellitus. ⁸ Antioxidants also had protective and anti-inflammatory impact in experimental uveitis models in animals. ^9,10 The Age-Related Eye Disease Study (AREDS) showed that daily high-dose intake of a combination of the antioxidants beta carotene, vitamins C and E, zinc, and copper resulted in a 25% reduction in the 5-year progression to late AMD, and this reduction was hypothesized to occur possibly by reducing ROS. ¹¹  

Recently, we showed in a standardized experimental model using lipopolysaccharide (LPS) in humans that systemic inflammation is associated with a diminished retinal vascular response to hyperoxia. ¹² This is most likely due to endothelial dysfunction induced by oxidative stress. ¹² In the present study we tested the hypothesis that the reduced response of retinal vascular reactivity to systemic hyperoxia during standardized experimental systemic inflammation can be restored by treatment with the AREDS combination of antioxidants. In addition, we measured the effects of the AREDS medication on retinal blood flow (RBF). 

Research Design and Methods

Subjects

The study protocol was approved by the Ethics Committee of the Medical University of Vienna and adhered to the guidelines set forth in the Declaration of Helsinki. Twenty-one healthy male volunteers between 18 and 35 years of age were included in this randomized (2:1 randomization), double masked, placebo-controlled study. All subjects signed written informed consent and passed a screening examination before the study day, including physical examination, venous blood sampling, assessment of visual acuity, slit lamp biomicroscopy, funduscopy, and measurement of intraocular pressure (IOP). Exclusion criteria were ametropia ≥3 D, other ocular abnormalities and any clinically relevant illness, blood donation, or intake of a medication, a vitamin or a mineral supplement in the 3 weeks before the study. Participants had to abstain from beverages containing alcohol or caffeine for 12 hours before each study day. 

Protocol

After instillation of 1 drop of tropicamide into the study eye and after a resting period of 15 minutes, RBF was assessed by combining measurements of retinal vessel diameters and laser-Doppler velocimetry (LDV). These values were taken as baseline values of the first study. The reactivity of retinal vessels and RBF to systemic hyperoxia was investigated during breathing of 100% oxygen (AGA Gases for Human Use, Vienna, Austria). Breathing of pure oxygen leads to vasoconstriction and reduction of RBF. ^13–16 For this purpose, an oxygen breathing period of 30 minutes was scheduled and retinal hemodynamic measurements were started 10 minutes after the start of breathing. 

Thereafter, intravenous infusion of a bolus containing 2 ng/kg bodyweight Escherichia coli endotoxin (U.S. Standard Reference Endotoxin; NIH-CC, Bethesda, MD) also known as lipopolysaccharide (LPS), was used on each study day as a standardized model of systemic inflammation and oxidative stress. LPS is a cell wall component of Gram-negative bacteria and a major mediator in the pathogenesis of septic shock. All measurements including the hyperoxia stimulus were repeated 4 hours after administration of LPS. 

After the first study day, subjects were randomly assigned to take either the AREDS medication (n = 14) or matching placebo (n = 7) for 14 days. Thereafter, a second study day with the same measurements and procedures as described for day 1 was performed. 

Study Medication

Subjects were given ocular vitamin supplements (Ocuvite PreserVision; Bausch & Lomb, Rochester, NY) containing β-carotene 28,640 IU, vitamin C (ascorbic acid) 452 mg, vitamin E (dl-α-tocopheryl acetate) 400 IU, zinc (zinc oxide) 69.6 mg, and copper (cupric oxide) 1.6 mg, with daily oral application divided into equal morning and evening doses. For blinding the tablets were divided and put into gelatin capsules; matching placebo capsules contained mannitol. 

Dynamic Vessel Analyzer (DVA)

The diameters of one major temporal retinal artery and vein within 1 to 2 disc diameters from the center of the optic disc were measured in mydriasis using the DVA (IMEDOS Systems, Jena, Germany). This system comprises a fundus camera (FF 450; Carl Zeiss Meditec AG, Jena, Germany), a high-resolution digital video camera, and a personal computer with analysis software. For the determination of retinal vessel diameters, recorded images are digitized and analyzed in real time with a frequency of 50 Hz. The system provides excellent reproducibility and sensitivity. ¹⁷ After selection of the measurement location, the DVA is able to follow the vessels during movements within the measurement window. 

Laser-Doppler Velocimetry

For measurement of retinal red blood cell velocity, we used a fundus camera–based system (LDV-5000; Oculix Inc., Arbaz, Switzerland). Measurements were performed in retinal veins at the same locations as diameter measurements. The principle of LDV is based on the optical Doppler effect. Laser light of a single-mode laser diode with a wavelength of 670 nm is scattered and reflected by moving erythrocytes leading to a broadened and shifted frequency spectrum. The frequency shift is proportional to the blood flow velocity in the retinal vessel. The maximum Doppler shift corresponds to the centerline erythrocyte velocity (V _max). ¹⁸ The Doppler-shift power spectra are recorded simultaneously for two directions of the scattered light. The scattered light is detected in the image plane of the fundus camera. This scattering plane can be rotated and adjusted in alignment with the direction of V _max, which enables absolute velocity measurements. From V _max, mean blood velocity in retinal vessels (V _mean) can be calculated as V _mean = V _max/2. Blood flow in the assessed retinal vein was calculated as Q = V _mean · D ²π/4, where V is velocity as assessed by LDV, and D is vessel diameter as measured with the DVA. 

Measurement of IOP and Systemic Hemodynamics

IOP was measured before and after DVA measurements, with a slit lamp–mounted Goldmann applanation tonometer (Haag-Streit, Bern, Switzerland). Before each measurement, 2 drops of oxybuprocainhydrochloride combined with sodium fluorescein were instilled for local anesthesia. 

Systolic, diastolic, and mean arterial blood pressures (SBP, DBP, MAP) were repeatedly measured before and after DVA measurements on the upper arm by an automated oscillometric device (HP-CMS patient monitor; Hewlett Packard, Palo Alto, CA). Pulse rate was automatically recorded by the same unit from a finger pulse oxymetric device. 

Statistical Analysis

Changes in retinal vessel diameters were expressed as percent (%) change over baseline values ± SD. Reactivity of retinal hemodynamic parameters to systemic hyperoxia was selected as the main outcome variable. Baseline parameters between groups were compared by analysis of variance (ANOVA). A repeated-measures ANOVA model was applied to detect statistically significant changes. P < 0.05 was considered as the level of significance. 

Results

Baseline parameters of both study groups are shown in Table 1. No significant differences were found between the two study groups. IOP was unaltered during all treatments in both groups. As expected, administration of LPS induced some side effects. These included temporary feeling of illness, increase in body temperature, and pulse rate (Table 1), shivering chills, headache, myalgia, sickness, and nausea, but were limited to a period of 8 hours after LPS infusion. Systemic blood pressure was slightly reduced after LPS in both groups and on both study days to a similar degree. On both study days LPS induced a pronounced increase in leukocyte counts (P < 0.003, Table 1) and C-reactive protein (CRP; P < 0.015) and a decrease in platelet counts (P < 0.007) 4 hours after administration. These effects were also not different between groups. 

Table 1.

 

Subject Characteristics and Parameters on Both Study Days under Baseline Conditions and 4 Hours after Administration of LPS

	AREDS Group (n = 14)	Placebo Group (n = 7)
Age, y	25.1 ± 3.4	25.4 ± 4.0
BMI, kg/m2	23.1 ± 2.0	23.5 ± 1.4

	Day 1		Day 15		Day 1		Day 15
	Baseline	LPS	Baseline	LPS	Baseline	LPS	Baseline	LPS
IOP, mm Hg	15.1 ± 4.3	14.4 ± 4.3	14.1 ± 3.6	15.1 ± 2.7	12.9 ± 2.3	13.1 ± 1.9	12.4 ± 1.9	12.9 ± 2.0
BT, °C	35.6 ± 0.4	36.7 ± 0.7	35.6 ± 0.5	36.7 ± 0.7	35.6 ± 0.4	36.5 ± 1.0	35.7 ± 0.6	36.6 ± 0.7
PR, min−1	66 ± 10	92 ± 11	65 ± 8	86 ± 10	62 ± 11	88 ± 16	60 ± 12	88 ± 10
MAP, mm Hg	82 ± 5	76 ± 4	83 ± 7	76 ± 6	81 ± 3	77 ± 7	78 ± 6	74 ± 5
LC, g/L	6.5 ± 1.8	9.6 ± 2.2	6.0 ± 1.6	10.0 ± 2.6	5.9 ± 0.8	10.0 ± 2.0	5.5 ± 0.8	10.5 ± 2.9
CRP, mg/dL	0.06 ± 0.04	0.12 ± 0.07	0.06 ± 0.03	0.14 ± 0.07	0.09 ± 0.07	0.22 ± 0.09	0.13 ± 0.15	0.19 ± 0.13
Platelets, g/L	221 ± 42	187 ± 45	224 ± 43	194 ± 40	229 ± 65	193 ± 61	227 ± 56	191 ± 67
Vit C, μmol/L	55.6 ± 12.6	14.8 ± 11.9	103.9 ± 12.7	72.3 ± 15.8	49.8 ± 10.5	39.2 ± 9.3	46.2 ± 10.6	36.0 ± 10.7
Vit E, μmol/L	25.3 ± 5.6	24.1 ± 5.0	48.2 ± 15.5	49.0 ± 15.2	26.8 ± 7.7	24.4 ± 6.4	24.5 ± 5.7	24.9 ± 5.0
Vit A, μmol/L	2.49 ± 0.46	2.33 ± 0.46	2.53 ± 0.51	2.39 ± 0.53	2.59 ± 0.69	2.31 ± 0.55	2.47 ± 0.48	2.36 ± 0.37
Artery, μm	135.6 ± 10.4	140.3 ± 10.7	134.5 ± 11.7	139.4 ± 11.9	129.5 ± 11.9	134.2 ± 12.9	133.0 ± 10.0	136.5 ± 9.5
Vein, μm	161.7 ± 15.5	168.4 ± 18.6	161.0 ± 16.4	167.7 ± 13.4	162.0 ± 10.6	168.5 ± 14.9	165.3 ± 14.7	169.6 ± 18.8

 BT, body temperature; PR, pulse rate; MAP, mean arterial pressure; LC, leukocyte count; CRP, C-reactive protein; Vit C, vitamin C; Vit E, vitamin E; Vit A, vitamin A.

Administration of the AREDS medication increased baseline vitamin C and E plasma levels compared to placebo (P < 0.001; Table 1). Whereas vitamin C levels were significantly reduced after LPS infusion on both study days in both groups (P < 0.013); vitamin E was not changed on either study day. The decrease in vitamin C on the second study day in the AREDS group was significantly greater than on day 1 (P < 0.001). This decrease was also more pronounced than in the placebo group (P < 0.001). There was no difference observed between the baseline vitamin A plasma levels on both study days in each group. Administration of LPS reduced vitamin A plasma levels on both study days to a comparable degree. 

LPS infusion induced a similar dilatation of retinal arteries on both study days (day 1: 3.5% ± 4.5% AREDS group, 3.7% ± 6.2% placebo group; day 15: 3.6% ± 4.1% AREDS group, 2.7% ± 2.6% placebo group; Table 1). There was no difference between groups on both study days. 

After administration of LPS on the first study day, we found a significantly reduced vasoconstriction of retinal arteries during systemic hyperoxia in both groups (Fig. 1, day 1; AREDS group: 12.5% ± 4.8% pre-LPS vs. 9.4% ± 4.6% post-LPS, P = 0.005; and placebo group: 9.2% ± 3.3% pre-LPS vs. 7.1% ± 3.5% post-LPS, P = 0.040). Retinal vein vasoconstriction was not diminished after LPS (AREDS: 13.5% ± 4.3% pre-LPS vs. 13.7% ± 5.3% post-LPS; and placebo: 13.5% ± 2.6% pre-LPS vs. 13.1% ± 3.2% post-LPS). The reduction of retinal blood velocity and RBF induced by 100% oxygen breathing was diminished after LPS in both groups (Fig. 2, day 1; velocity—AREDS group: 34.1% ± 8.7% pre-LPS vs. 26.5% ± 11.9% post-LPS, P = 0.001; placebo group: 38.9% ± 9.9% pre-LPS vs. 28.3% ± 11.3% post-LPS, P = 0.035; flow—AREDS: 50.4% ± 8.9% pre-LPS vs. 44.9% ± 11.6% post-LPS, P = 0.003; and placebo: 54.2% ± 8.6% pre-LPS vs. 46.0% ± 6.9% post-LPS, P = 0.035). 

Figure 1.

 

Vessel response to breathing of 100% oxygen before and after 14 days of AREDS medication versus placebo.

Figure 2.

 

Blood velocity and blood flow response to breathing 100% oxygen before and after 14 days of AREDS medication versus placebo.

On the second study day, after treatment with AREDS medication, arterial vasoconstriction during hyperoxia was sustained after LPS (Fig. 1, day 2; 13.1% ± 4.5% pre-LPS vs. 13.1% ± 5.0% post-LPS, P = 0.992). In the placebo group, however, the oxygen-induced arterial vasoconstriction after LPS was again diminished (11.2% ± 4.2% pre-LPS vs. 7.4% ± 4.2% post-LPS, P = 0.059). As also observed on the first study day venous diameter reactivity was unaltered by either the AREDS medication or LPS (AREDS group: 13.1% ± 4.1% pre-LPS vs. 14.5% ± 5.0% post-LPS; placebo group: 13.6% ± 2.0% pre-LPS vs. 11.7% ± 2.4% post-LPS). Venous reactivity was comparable between AREDS and placebo. Similar to arterial diameter response, the reactivity of RBF parameters after LPS was sustained on the second study day in the group taking the AREDS medication, but was significantly diminished in the placebo group (Fig. 2, day 2; velocity—AREDS group: 37.3% ± 11.3% pre-LPS vs. 35.4% ± 10.1% post-LPS, P = 0.621; placebo group: 36.0% ± 13.7% pre-LPS vs. 28.2% ± 11.4% post-LPS, P = 0.036; flow—AREDS: 52.8% ± 10.5% pre-LPS vs. 52.4% ± 10.5% post-LPS, P = 0.902; and placebo: 52.4% ± 9.3% pre-LPS vs. 44.2% ± 6.3% post-LPS, P = 0.008). 

Discussion

Our findings support previous data showing that the inflammatory response after administration of LPS induces reduced retinal vasoconstriction during hyperoxia. ⁸ Most likely, this is due to impaired endothelial function during systemic inflammation. ¹⁹ It has been shown that LPS leads to increased oxidative stress and reduced endothelial function in other vascular beds. ^20,21 The mechanisms lying beneath endothelial dysfunction during inflammatory processes have not yet been fully elucidated. ROS have been identified as damaging endothelial cells and enhancing microvascular permeability during inflammation and may therefore play a key role in this process. ²² This concept is also supported by the results of the present study. Plasma levels of vitamin C, acting as a main scavenger for ROS, significantly decreased during LPS indicating increased vitamin C consumption. Hence, the data of the present study suggest that during systemic inflammation, a considerable amount of ROS was produced. 

The normalization of RBF reactivity to hyperoxia during experimental inflammation after 2 weeks of intake of highly dosed antioxidants indicates that endothelial dysfunction at the level of the retinal vessel due to systemic inflammation can be reduced by the use of the AREDS medication. It is not known to what extent the endothelium-derived substances mediate retinal vascular vasoconstriction during systemic hyperoxia. Results in animal retina indicate a role of the arachidonic acid metabolites thromboxane and 20-hydroxyeicosatetrienoic acid ²³ and also of endothelin. ²⁴ Experiments with endothelin-receptor antagonists in humans have also demonstrated that the vascular response to hyperoxia is dependent on endothelin-1 indicating a major role of the endothelium. ²⁵  

There is some evidence indicating endothelial dysfunction in AMD. ²⁶ Changes in AMD may fairly well be associated with endothelium disturbances, because vascular endothelial growth factor, which is one of the most important mediators of this disease, is mainly produced in the endothelium and has been found elevated in dry as well as in wet AMD. ²⁶  

The composition of the AREDS medication has been questioned in the recent years, particularly because of the high concentration of some of the components. High levels of β-carotene have been found to raise the risk of lung cancer in smokers ²⁷ and vitamin E has been associated with an increased risk of heart failure in people with vascular disease or diabetes, ²⁸ limiting the use in these subgroups of patients with AMD. As a consequence, many different combinations of dietary supplements are currently marketed for the use in patients with AMD, some with reduced concentrations of vitamins C and E and many of them omitting β-carotene. Evidence of therapeutic efficacy is available, however, only for the original composition of the AREDS medication, and little is known about types and amounts of antioxidants that are sufficient to reduce AMD progression. ²⁹ In addition lutein and zeaxanthin, the natural components of the macular pigments, have been proposed as potential dietary supplements for AMD, because of their oxygen scavenger properties and their ability to absorb high-energy blue light. Recently, lutein has been shown to have anti-inflammatory effects in an LPS-induced model of ocular inflammation in rats. ³⁰ Another focus was directed toward omega-3 free fatty acids, which have potent anti-inflammatory properties. In keeping with this hypothesis, eicosapentaenoic acid-rich diet resulted in significant suppression of neovascularization and inflammatory molecules in a mouse model of choroidal neovascularization. ³¹ Reduced intake of lutein/zeaxanthin and omega-3 free fatty acids appears to be related to an increased risk of AMD. ^32,33 Evidence that intervention with lutein/zeaxanthin or omega-3 free fatty acids reduces the progression of AMD is lacking, however. 

Hence, the question of dosage as well as the optimal composition of a dietary supplement to retard AMD is far from being solved. ²⁹ Long-term, randomized, controlled studies proving the efficacy of all the different dosages and compositions of dietary supplements do not seem feasible. The model presented herein may be a practical approach to testing the antioxidant effect of different supplements and different dose regimen in the human eye. 

In conclusion, this study confirms previous data showing that the inflammatory response after administration of LPS induces impaired endothelial function. Oxidative stress appears to play a role in endothelial dysfunction due to inflammatory processes. Antioxidants seem to reduce oxidative stress and endothelial dysfunction by eliminating ROS in the applied model of systemic inflammation leading to a sustained vasoconstrictor response of retinal vessels to 100% oxygen breathing. Our model may be an attractive approach to the study of the antioxidative capacity of different supplementations and their dosing in the human retina. 

Footnotes

 Disclosure: B. Pemp, None; E. Polska, None; K. Karl, None; M. Lasta, None; A. Minichmayr, None; G. Garhofer, None; M. Wolzt, None; L. Schmetterer, None