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Astaxanthin Decreases Inflammatory Biomarkers Associated with Cardiovascular Disease in Human Umbilical Vein Endothelial Cells

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Oxidative stress and inflammation are strongly linked to the development of cardiovascular disease (CVD). As a potent antioxidant, astaxanthin may downregulate inflammation-associated factors involved in endothelial dysfunction and CVD progression. We studied the possible protective effects of astaxanthin against endothelial dysfunction in human umbilical vein endothelial cells (HUVEC) induced with hydrogen peroxide (H2O2). Cells were pre-incubated with 0, 0.01, 0.1, or 1.0 μmol/L astaxanthin for 48 h, then oxidative stress induced with 100 μmol/L H2O2 overnight. Uptake kinetics of astaxanthin showed a time-dependent uptake of astaxanthin (1.0 μmol/L) by HUVEC over the 48 h incubation period. Oxidative stress induction with H2O2 in HUVEC decreased intracellular antioxidant activity and increased the production of inflammatory biomarkers and reactive oxygen species (ROS). In contrast, pre-incubation of HUVEC with astaxanthin prior to H2O2 stress increased (P < 0.05) superoxide dismutase activity and decreased ROS, prostaglandin E2, leukotriene B4, NO, IL-8, and IFN- production. Pre-treatment with astaxanthin also downregulated the transcriptional activation of NF-κB and activator protein-1, thereby inhibiting downstream production of inflammatory mediators and cytokines. Therefore, astaxanthin protects against factors initiated by H2O2 addition, likely by scavenging ROS required for transcriptional activation and inhibiting the production of inflammatory biomarkers involved in endothelial dysfunction and CVD. Abbreviations: AP-1: activator protein-1; CVD: cardiovascular disease; CS: culture supernatant; EGM-2: endothelial growth medium-2; GPx: glutathione peroxidase; H2O2: hydrogen peroxide; HUVEC: human umbilical vein endothelial cell; LTB4: leukotriene B4; PGE2: prostaglandin E2; ROS: reactive oxygen species; SOD: superoxide dismutase; THF: tetrahydrofuran.
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Columbia International Publishing
American Journal of Advanced Food Science and Technology
(2013) 1: 1-17
doi:10.7726/ajafst.2013.1001
Research Article
______________________________________________________________________________________________________________________________
*Corresponding e-mail: boonchew@wsu.edu
1* School of Food Science, Washington State University, Pullman, WA 99164-6376
2 School of Molecular Biosciences, Washington State University, Pullman, WA 99164-4234
1
Astaxanthin Decreases Inflammatory Biomarkers
Associated with Cardiovascular Disease in Human
Umbilical Vein Endothelial Cells
Weslee Chew1, Bridget D. Mathison1, Lindsey L. Kimble1, Philip F. Mixter2, and Boon P. Chew1*
Received 24 November 2012; Published online 12 January 2013
© The author(s) 2013. Published with open access at www.uscip.org
Abstract
Oxidative stress and inflammation are strongly linked to the development of cardiovascular disease (CVD). As
a potent antioxidant, astaxanthin may downregulate inflammation-associated factors involved in endothelial
dysfunction and CVD progression. We studied the possible protective effects of astaxanthin against
endothelial dysfunction in human umbilical vein endothelial cells (HUVEC) induced with hydrogen peroxide
(H2O2). Cells were pre-incubated with 0, 0.01, 0.1, or 1.0 μmol/L astaxanthin for 48 h, then oxidative stress
induced with 100 μmol/L H2O2 overnight. Uptake kinetics of astaxanthin showed a time-dependent uptake of
astaxanthin (1.0 μmol/L) by HUVEC over the 48 h incubation period. Oxidative stress induction with H2O2 in
HUVEC decreased intracellular antioxidant activity and increased the production of inflammatory biomarkers
and reactive oxygen species (ROS). In contrast, pre-incubation of HUVEC with astaxanthin prior to H2O2 stress
increased (P < 0.05) superoxide dismutase activity and decreased ROS, prostaglandin E2, leukotriene B4, NO,
IL-8, and IFN- production. Pre-treatment with astaxanthin also downregulated the transcriptional activation
of NF-κB and activator protein-1, thereby inhibiting downstream production of inflammatory mediators and
cytokines. Therefore, astaxanthin protects against factors initiated by H2O2 addition, likely by scavenging ROS
required for transcriptional activation and inhibiting the production of inflammatory biomarkers involved in
endothelial dysfunction and CVD.
Abbreviations: AP-1: activator protein-1; CVD: cardiovascular disease; CS: culture supernatant; EGM-2:
endothelial growth medium-2; GPx: glutathione peroxidase; H2O2: hydrogen peroxide; HUVEC: human
umbilical vein endothelial cell; LTB4: leukotriene B4; PGE2: prostaglandin E2; ROS: reactive oxygen species;
SOD: superoxide dismutase; THF: tetrahydrofuran.
Keywords: Astaxanthin; HUVEC; Inflammation; Cardiovascular disease
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1. Introduction
Cardiovascular disease (CVD) is the leading cause of death for both men and women in the United
States, with more than 600,000 deaths attributed to CVD in 2006 (Lloyd-Jones et al., 2010). Certain
conditions and lifestyle factors can greatly increase ones risk of developing CVD. While the
relationships between these factors are complex, oxidative stress and inflammation are strongly
linked to the development of CVD (Lee et al., 2011a). Despite this connection, there are few
therapeutic interventions that successfully address this relationship between oxidative stress and
CVD (Pashkow et al., 2008).
Oxidative stress results from the overproduction of reactive oxygen species (ROS). These highly
reactive molecules cause cellular damage, including apoptosis, protein oxidation, DNA modification
and lipid peroxidation (Chew and Park, 2004). The endothelial cells lining the blood vessels are
very sensitive to injury caused by oxidative stress. Damage to endothelial cell structure and
function contributes to blood vessel diseases, including atherosclerosis, thrombosis and vasculitis
(Hafizah et al., 2010). Additionally, ROS activate transcriptional messengers, such as NF-κB and
activator protein-1 (AP-1), both of which upregulate the production of inflammatory cytokines and
mediators. Oxidative stress and inflammation resulting from the production of these inflammatory
molecules are implicated in endothelial dysfunction and the subsequent development of CVD
(Foncea et al., 2000).
ROS are natural byproducts of cellular respiration and are essential for cell signaling and
homeostasis. Under normal conditions, ROS concentrations are tightly controlled by endogenous
antioxidant systems, including superoxide dismutase (SOD), glutathione peroxidase (GPx), and
catalase. However, when endogenous antioxidants are overwhelmed by the overproduction of ROS,
oxidative stress occurs resulting in inflammation and cellular damage (Lee et al., 2011a), thereby
requiring dietary antioxidants to help scavenge these harmful molecules. Therefore, dietary
antioxidants may play a beneficial role in treating CVD as they scavenge ROS and slow the
progression of oxidative damage. Astaxanthin, a keto oxycarotenoid, is a potent antioxidant; its
antioxidant activity has been reported to be higher than that of β-carotene, α-carotene, and lutein
(Naguib, 2000). Astaxanthin modulates oxidative stress and inflammatory mediators, and has
shown to be beneficial against multiple disease models (Fassett and Coombes, 2011). Astaxanthin
inhibited NO production and inflammatory gene expression by suppressing NF-κB activation in
lipopolysaccharide-stimulated RAW264.7 macrophage cell lines (Lee et al., 2003). Astaxanthin also
suppressed serum NO and other inflammatory mediators in lipopolysaccharide-treated mice.
Additionally, significant cardioprotection has been demonstrated with astaxanthin in multiple
animal models of ischemia-reperfusion, with up to 70% protection from ischemic damage
(Pashkow et al., 2008).
Endothelial cells play a crucial role in many vascular functions, including cell adhesion,
inflammatory responses, regulation of permeability, and vasoactivity. Therefore, human umbilical
vein endothelial cells (HUVEC) are commonly used to study vascular dysfunction (Hafizah et al.,
2010). Further, hydrogen peroxide (H2O2) is a principal mediator of ROS-dependent signaling, and
exposure of endothelial cells to H2O2 induces the activation of ROS-dependent signaling cascades
(Eligini et al., 2009). Incubation of HUVEC with 100 μmol/L H2O2 has been shown to induce
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oxidative stress, without inducing apoptosis (Lin et al., 2011), by inhibiting endogenous antioxidant
activity and activating inflammatory signaling pathways (Hafizah et al., 2010).
In summary, the ability of exogenous antioxidants to scavenge ROS, such as H2O2 and NO, and
downregulate gene activation associated with the overproduction of inflammatory mediators and
cytokines has been established. The objective of this study is to assess the protective effect of
astaxanthin on inflammatory response in HUVEC induced with H2O2.
2. Materials and Methods
2.1 Cell culture and carotenoid preparation
HUVEC (ATCC, Manassas, VA) were cultured in EGM-2 supplemented with 100 units/mL penicillin
G, 100 µg/mL streptomycin sulfate, 0.25 µg/mL amphotericin B, and 10% newborn calf serum
(HyClone, Logan, UT) at 37°C in a humidified 5% CO2 atmosphere. Cell culture medium and all other
chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. Adherent
cells were detached with trypsin-EDTA (0.25% trypsin-0.05% EDTA in Hanks’ balanced salt
solution without Mg2+ and Ca2+) and cell number enumerated using a Z1 Coulter Particle Counter
(Beckman Coulter, Brea, CA). All experiments were conducted using cells within 2 passage
numbers. Immediately prior to use, astaxanthin was first solubilized in tetrahydrofuran (THF),
gradually added to newborn calf serum while mixing, and the mixture slowly added to EGM-2 (final
THF concentration was 0.1%) while mixing. The use of THF is an acceptable medium for dissolving
carotenoids in cell culture (Bertram et al., 1991).
2.2 Kinetic carotenoid uptake study
Cells were suspended in medium containing 1.0 μmol/L astaxanthin, plated at 5 105 cells/well in
6 well culture plates and incubated for 0, 6, 12, 24, or 48 h (n = 6) to study astaxanthin uptake by
HUVEC. At the end of each incubation period, cells were dissociated with trypsin-EDTA and lysed
with 1 mL buffer containing 100 mM sucrose, 1 mM ethylene glycol tetraacetic acid, 20 mM 3-(N-
morpholino)propanesulfonic acid (pH 7.4), and 0.1% bovine serum albumin. The hydrophobic
fraction containing the carotenoid was sequentially extracted with 3 mL acetone containing 0.1%
butylated hydroxytoluene followed by 3 mL hexane:ethyl acetate (1:1; v:v). After centrifugation, the
organic layer was collected, dried under nitrogen gas, and the residue dissolved in a mixture of
hexane:acetone (82:18; v:v), the mobile phase used in the reverse phase HPLC separation (Alliance
2690, Waters, Milford, MA). Samples were eluted through a 3 μm silica column (150 x 4.6 mm, Luna,
Phenomenex, Torrance, CA), and absorbance monitored at 474 nm (Photodiode Array Detector
996, Waters, Milford, MA).
2.3 Inflammatory biomarker assays
Cells were suspended in treatment medium supplemented with 0, 0.01, 0.1, or 1.0 μmol/L
astaxanthin (n = 6) and plated at 5 105 cells/well in 6 well plates. After culturing for 48 h,
oxidative stress was induced with 100 μmol/L H2O2 and the cells incubated for an additional 18 h.
Cultures containing no H2O2 served as a negative control. The resultant culture supernatant (CS)
and cell pellet were collected and stored at -80°C until further analysis. Prostaglandin E2 (PGE2) and
leukotriene B4 (LTB4) were analyzed in CS by ELISA (Parameter PGE2, Parameter LTB4, R&D
Systems, Minneapolis, MN). The lower limits of detection were 30.9 and 27.6 pg/mL for PGE2 and
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LTB4, respectively. NO was measured in CS by colorimetric assay with a lower limit of detection of
2.5 μmol/L (Nitrate/Nitrite Colorimetric Assay Kit, LDH method, Cayman Chemical, Ann Arbor, MI).
Pro-inflammatory cytokines IL-1α, IL-1β, IL-2, IL-6, IL-8, IFN-γ, and TNF-α and anti-inflammatory
cytokines IL-4 and IL-10 were analyzed in CS by chemiluminescent array ELISA (Quansys Q-Plex
Cytokine Array, Logan, UT); data was analyzed using Quansys Q-View 2.5.2 software. The lower
limit of detection was ≤ 1.0, 3.24, 2.76, 1.78, 3.81, ≤ 1.0, ≤ 1.0, 1.0, and ≤ 1.0 pg/mL for IL-1α, IL-
1β, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ and TNF-α. In addition, IL-17 was analyzed in CS by ELISA
(Human IL-17 Quantikine ELISA Kit, R&D Systems, Minneapolis, MN); the lower limit of detection
for this assay was ≤ 15 pg/mL.
2.4 Antioxidant activity
Cell pellets were analyzed for GPx and SOD activity. Cell extracts were prepared by solubilizing the
cell pellets in sonication buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.2,
1 mM ethylene glycol tetraacetic acid, 210 mM mannitol, 70 mM sucrose) and sonicated 5 times
with 2 sec pulses (Branson Sonifier, Danbury, CT) at 70% maximum setting. Cellular GPx activity
was measured using a colorimetric assay (BIOXYTECH cGPX-340, OxisResearch, Foster City, CA)
monitoring change in absorbance at 340 nm (μQuant, BioTek Instruments, Winooski, VT); the
detection limit was 5.6 mU/mL GPx enzyme activity. Cellular SOD activity was also measured with a
colorimetric assay, with a detection limit of 0.025 U/mL SOD (Superoxide Dismutase Assay, Cayman
Chemical, Ann Arbor, MI).
2.5 Transcription factor analysis
The cell pellets were analyzed to determine NF-κB activity by ELISA (NF-κB human p50
Transcription Factor Assay, Cayman Chemical, Ann Arbor, MI). Nuclear extracts were prepared
according to manufacturer directions with one modification: nuclear pellets were resuspended in
30 μL of extraction buffer. Protein concentrations in nuclear extracts were determined using a
colorimetric assay (Micro BCA Protein Assay Kit, Pierce, Rockford, IL). After standardizing NF-κB
activity using nuclear protein concentrations, results were expressed as % response compared to
the positive control. To further elucidate the mechanism of action, an additional set of nuclear
extracts (n = 6) were prepared using an alternate method (Nuclear Extraction Kit, Panomics,
Freemont, CA) for AP-1 binding activity. HUVEC were cultured with 0 or 0.1 μmol/L astaxanthin for
48 h and then oxidative stress induced with 100 μmol/L H2O2, as previously described. AP-1
binding activity in these extracts was determined using an indirect capture ELISA (AP-1
Transcription Factor Kit, Panomics, Freemont, CA). Results were expressed as % response
compared to the positive control.
2.6 ROS production
ROS production was determined using carboxy-H2DCFDA, a fluorescent marker for ROS in live cells
(Image-iT LIVE Green Reactive Oxygen Species Detection Kit, Molecular Probes, Eugene, OR). To
determine acute ROS production in response to H2O2 induction, cells were plated at 5 104
cells/well in 96-well plates and cultured with 0 or 0.1 μmol/L astaxanthin (n = 6), as described
earlier. Cells were washed with Hanks’ balanced salt solution, 25 μmol/L carboxy-H2DCFDA added,
incubated for 30 min at 37C then washed again with Hanksbalanced salt solution. Following the
addition of 100 μmol/L H2O2, fluorescence was measured every 30 min for 2 h (ex 495 nm/em 529
nm) in a fluorescent plate reader (FLx800, BioTek Instruments, Winooski, VT). An additional
culture containing no H2O2 served as a negative control.
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Chronic ROS response was measured in a similar manner, following an incubation of cells with H2O2
for 18 h.
2.7 Statistical analysis
Data were analyzed by ANOVA using the General Linear Model procedure and treatment means
were compared using protected LSD. Probability values of P < 0.05 were considered statistically
significant.
3. Results
3.1 Carotenoid uptake
Astaxanthin accumulated in a time-dependent manner in HUVEC incubated with 1.0 µmol/L
astaxanthin. There was significant uptake at 6 h, with a maximum concentration of 125 ± 5
pmol/106 cells observed at 48 h (Fig. 1). Astaxanthin was undetectable in parallel cultures not
supplemented with astaxanthin.
Fig 1. Accumulation of astaxanthin in HUVEC incubated with 1.0 mol/L astaxanthin for 0, 6, 12, 24,
and 48 h. Values are means ± SEM.
3.2 Inflammation biomarkers.
Following stress with H2O2, HUVEC showed increased (P < 0.05) production of PGE2, LTB4, and NO
when compared to negative controls (Table 1). PGE2 production was dramatically reduced (P <
0.05) in cultures incubated in 0.01, 0.1, as well as 1.0 μmol/L astaxanthin, compared to
corresponding positive control cultures, with levels generally similar to those of the negative
control cultures. Production of LTB4 was reduced (P < 0.05) 40-65% when cells were incubated
with astaxanthin compared to cultures without astaxanthin. NO production was similarly decreased
(P < 0.05) approximately 25% in the presence of 0.1 and 1.0 μmol/L astaxanthin.
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Table 1 Production of inflammatory biomarkers in HUVEC cultures pre-incubated in the presence
of 0, 0.01, 0.1 and 1.0 µmol/L astaxanthin and subsequently stressed with 100 μmol/L H2O2. Data
are presented as means SEM. *Labeled means in a row represent differences compared to the
H2O2-stimulated control, P < 0.05.
Astaxanthin (μmol/L)
0
0.01
0.1
No H2O2
------------------------------ H2O2 ------------------------------
PGE2 (pg/μg protein)
2.01 ± 0.56*
20.28 ± 2.66
1.26 ± 0.44*
1.54 ± 0.50*
LTB4 (pg/mg protein)
119 ± 37*
955 ± 84
581 ± 122*
328 ± 32*
NO (nmol/μg protein)
0.49 ± 0.01*
2.30 ± 0.23
2.01 ± 0.14
1.65 ± 0.03*
Incubation of HUVEC with H2O2 in positive control cultures stimulated (P < 0.05) the production of
the pro-inflammatory cytokines IL-2, IL-6, IL-8, IL-17, IFN-γ and TNF-α (Table 2). The presence of
astaxanthin in these cultures had mixed effects on these inflammatory cytokines. At all
concentrations of astaxanthin, IL-8 production decreased (P < 0.05), whereas only 0.1 μmol/L
astaxanthin significantly inhibited (P < 0.05) IFN-γ production. Pre-incubation of HUVEC with
astaxanthin had no effect on IL-2, IL-6 and TNF-α. Interestingly, pre-incubation with 0.01 μmol/L
astaxanthin further increased (P < 0.05) IL-17 production. IL-1α and IL-concentrations were not
significantly different between controls and astaxanthin treatment cultures (overall means ± SE
were 0.27 ± 0.06 and 0.71 ± 0.10 pg/μg protein, respectively).
Table 2 Production of cytokines in HUVEC cultures pre-incubated in the presence of 0, 0.01, 0.1 and
1.0 µmol/L astaxanthin and subsequently stressed with 100 μmol/L H2O2. Data are presented as
means SEM. *Labeled means in a row represent differences compared to the H2O2-stimulated
control, P < 0.05.
Astaxanthin (μmol/L)
0
0.01
0.1
1.0
Cytokine (pg/μg protein)
No H2O2
------------------------------ H2O2 ------------------------------
IL-2
0.29 ± 0.04*
1.52 ± 0.42
1.32 ± 0.38
0.84 ± 0.07
0.84 ± 0.12
IL-6
15.1 ± 0.9*
39.3 ± 4.5
37.9 ± 2.7
34.4 ± 2.9
43.7 ± 5.1
IL-8
29.2 ± 1.1*
136.4 ± 13.8
82.2 ± 7.0*
79.3 ± 5.0*
96.7 ± 4.0*
IL-17
0.93 ± 0.07*
1.89 ± 0.21
6.43 ± 0.60*
3.88 ± 0.53
3.48 ± 1.07
IFN-γ
0.05 ± 0.02*
0.20 ± 0.04
0.13 ± 0.04
0.09 ± 0.04*
0.19 ± 0.05
TNF-
0.47 ± 0.12*
1.35 ± 0.32
1.15 ± 0.18
1.11 ± 0.25
1.26 ± 0.16
IL-4
0.15 ± 0.02*
0.45 ± 0.10
0.31 ± 0.06
0.38 ± 0.05
0.38 ± 0.06
IL-10
0.13 ± 0.01*
0.51 ± 0.04
0.48 ± 0.04
0.50 ± 0.08
0.51 ± 0.07
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Incubation of HUVEC with H2O2 in positive control cultures also stimulated (P < 0.05) the
production of the anti-inflammatory cytokines IL-4 and IL-10 (Table 2). However, astaxanthin had
no effect on the production of these anti-inflammatory cytokines compared to the positive control.
3.3 Antioxidant activity.
SOD activity in HUVEC was inhibited (P < 0.01) by H2O2 when compared to cells cultured without
H2O2; however, pre-incubation with astaxanthin increased (P < 0.05) SOD activity in a dose-
dependent manner (Fig. 2). GPx activity also decreased (P < 0.01) in the presence of H2O2, with
concentrations averaging 0.15 ± 0.01 and 0.01 ± 0.01 mU/µg protein in the negative and positive
control cultures, respectively. Pre-incubation with astaxanthin had no effect on GPx activity (overall
treatment mean ± SE, 0.01 ± 0.01 mU/µg protein).
Fig 2. SOD activity (mean ± SEM) in HUVEC pre-incubated with 0, 0.01, 0.1, or 1.0 µmol/L
astaxanthin for 48 h and subsequently stressed with 100 μmol/L H2O2. An additional culture
containing no H2O2 served as a negative control. *Significantly different from positive control (P <
0.05).
3.4 Transcription factor activation.
NF-κB p50 activity was stimulated (P < 0.01) in HUVEC incubated in the presence of H2O2 when
compared to cells cultured without H2O2 (Fig. 3). However, pre-incubation with astaxanthin
decreased (P < 0.05) nuclear p50 activity by 30-40%. Similarly, AP-1 activity also increased (P <
0.01) in the presence of H2O2 while pre-incubation with 0.1 µmol/L astaxanthin dramatically
inhibited (P < 0.05) AP-1 activity (Fig. 4).
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Fig 3. NF-κB p50 activity (mean ± SEM) by HUVEC pre-incubated with 0, 0.01, 0.1, or 1.0 µmol/L
astaxanthin for 48 h and subsequently stressed with 100 μmol/L H2O2. An additional culture
without H2O2 served as the negative control. Differences in p50 activity (expressed as percent
response) were calculated by dividing the optical density (OD) of each treatment sample by the OD
of the positive control. *Significantly different from positive control (P < 0.05).
Fig 4. AP-1 activity (mean ± SEM) by HUVEC cells pre-incubated with 0.1 µmol/L astaxanthin for 48
h and subsequently stressed with 100 μmol/L H2O2. An additional culture containing no H2O2
served as a negative control. Differences in AP-1 (expressed as percent response) were calculated
by dividing the optical density (OD) of each treatment sample by the OD of the positive control. *
Significantly different from positive control (P < 0.05).
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3.5 ROS production.
While ROS production by HUVEC was very low in the absence of H2O2, acute ROS production
increased (P < 0.05) dramatically and linearly in H2O2-stimulated cultures over the 120 min period
studied (Fig. 5). Pre-incubation with 0.1 μmol/L astaxanthin decreased (P < 0.05) ROS production;
however, ROS concentrations in the presence of astaxanthin were higher that observed in cultures
not stimulated with H2O2. Similarly, ROS production increased (P < 0.01) in positive control cultures
after overnight stimulation with H2O2. Pre-incubation with 0.1 μmol/L astaxanthin decreased (P <
0.05) ROS production by 44% as compared to the positive control (data not shown).
Fig 5. Acute ROS production (mean ± SEM) by HUVEC pre-incubated with 0.1 µmol/L astaxanthin
for 48 h and loaded with a 25 mol/L carboxy-H2DCFDA working solution for 30 min at 37°C. Cells
were stimulated with 100 μmol/L H2O2 and fluorescence intensity (FI) was measured every 30 min
(excitation 495 nm/ emission 529 nm). An additional culture containing no H2O2 served as a
negative control. *Significantly different from positive control (P < 0.05).
4. Discussion
The high antioxidant and anti-inflammatory activity of astaxanthin is largely attributed to its
precise transmembrane alignment in the lipid bilayer of cellular membranes; the presence of polar
ionone rings at both ends of the non-polar conjugated carbon chain allows the astaxanthin molecule
to span the polar-nonpolar-polar lipid bilayer. In addition to preventing lipid-based oxidation, this
alignment exposes the ionone rings to interact with ROS in the aqueous environment and likely
provides proximity to cofactors, such as vitamin C (Pashkow et al., 2008). We previously
demonstrated that astaxanthin administered orally is taken up in significant amounts by
subcellular organelles of canine and feline lymphocytes, thus showing specific uptake of the
carotenoid by target cells (Park et al., 2010). Kinetic study of astaxanthin accumulation by HUVEC
showed significant, albeit low, uptake at 6 h, with maximal concentration observed at 48 h.
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Astaxanthin accumulation in HUVEC protects these cells against H2O2-induced oxidative stress by
triggering a number of modifications in the production of inflammatory biomarkers.
Oxidative stress was successfully induced in HUVEC cultures with 100 μmol/L H2O2, as evident
from suppressed SOD and GPx activities, and the stimulation of several biomarkers including PGE2,
LTB4, NO, IL-2, IL-6, IL-8, IL-17, IFN-γ, TNF-α, NF-κB, and AP-1. Pre-incubation of HUVEC with
astaxanthin restored SOD activity, indicating antioxidative action by astaxanthin; but it had no
significant effect on GPx activity suggesting different actions on these two endogenous antioxidants.
Cytosolic GPx in endothelial cells has been shown to be inactivated by mediators of inflammatory
response, including superoxide anion, hypochlorous acid, and NO (Moutet et al., 1998). Resveratrol
increased both SOD and GPx activity in response to DMNQ-induced oxidative stress in HUVEC
(Spanier et al., 2009).
Induction of oxidative stress in HUVEC is further evidenced by the dramatic increase in ROS
production in response to an acute stimulation by H2O2, and the continued elevation after 18 h H2O2
exposure. Pretreatment with astaxanthin decreased ROS production in response to both acute and
long term stimulation with H2O2. Resveratrol, epigallocatechin gallate, quercetin, and a combination
of simvastatin and nifedipine also have been shown to reduce ROS production in HUVEC stimulated
with H2O2 (Choi et al., 2005; Chen et al., 2010; Kao et al., 2010). Therefore, stimulation with H2O2
suppressed antioxidant function, increased ROS accumulation, and induced oxidative stress.
However, pretreatment with astaxanthin alleviated this oxidative stress by restoring SOD activity
and clearing intracellular ROS.
ROS act as second messengers activating transcription factors, such as NF-κB and AP-1, that
upregulate the production of inflammatory cytokines and mediators. NF-κB plays a critical role in
inflammation associated with the progression of atherosclerosis and is often referred to as the
“master switch” of inflammatory response. NF-κB exists in the cytosol in an inactive form bound to
an inhibitor IκB, and following inflammatory stimuli, intracellular signals activate the IκB kinase
complex, in turn inducing phosphorylation and degradation of IκB from NF-κB. Activation of the
NF-κB pathway results in the nuclear appearance of p50 and p65, and the subsequent
transcriptional induction of genes associated with the production of inflammatory mediators and
cytokines (Kim et al., 2008). While several ROS play a role in the activation of NF-κB, H2O2
specifically modulates NF-κB by promoting the activation of the NIK/IKK pathway (Kim et al.,
2008). HUVEC incubated in the presence of H2O2 showed an increase in nuclear p50 activity,
indicating the upregulation of the NF-κB pathway. However, pretreatment with astaxanthin
decreased nuclear p50 activity. Similarly, a purified extract of herbs commonly used for treatment
of inflammatory diseases in Asia, also was shown to downregulate NF-κB activation in HUVEC
stimulated with 10 ng/mL TNF-α (Mo et al., 2007).
In addition to its NF-κB activity, H2O2 increases the activity of the serine-threonine kinases of the
MAPK family. The p38 and JNK pathways have numerous effects, but one common target is the
activation of AP-1. JNKs are termed stress-activated protein kinases since many of the JNK
activators can be regarded as cellular stress (Lo et al., 1996). HUVEC incubated in the presence of
H2O2 also showed an upregulation of AP-1; in contrast, pretreatment with astaxanthin decreased
AP-1 activity. The ability of antioxidants to downregulate NF-κB and AP-1 is likely due to their
Weslee Chew, Bridget D. Mathison, Lindsey L. Kimble, Philip F. Mixter, and Boon P. Chew / American Journal of
Advanced Food Science and Technology (2013) 1: 1-14
11
potent scavenging of ROS, inhibiting ligands required for downstream transcriptional activation
and inflammatory gene expression.
The activation of NF-κB and AP-1 leads to the production of inflammatory mediators and cytokines.
Oxidative stress induction with H2O2 increased the production of PGE2, LTB4, and NO in HUVEC.
However, pre-incubation with astaxanthin decreased the production of these inflammatory
mediators. In addition to NO, the arachidonic acid metabolites (PGE2 and LTB4) are key markers of
endothelial integrity. In fact, inflammatory activation of endothelial cells and the subsequent
release of these mediators has been associated with the development of atherosclerosis,
hypertension and heart failure (Olszanecki et al., 2006).
While H2O2 stimulation is shown to directly upregulate NO production in HUVEC, some studies
suggest a protective effect of this NO over-production (Liu et al., 2009). NO is an important
regulator of cardiovascular homeostatsis that is synthesised by endothelial NO synthase (eNOS) in
the vasculature. H2O2 has been shown to induce eNOS gene expression and increase NO production
in endothelial cells; this may represent an initial acute compensatory response to increased stress
intended to protect the cells rather than produce cell injury (Cai et al., 2003). However, as
endogenous antioxidant defenses become overwhelmed by ROS, oxidative stress and inflammation
ensues, leading to cellular damage. Pro-inflammatory cytokines, such as TNF-α, have been shown
to upregulate inducible NO synthase (iNOS) activity and NO production in HUVEC (Xia et al., 2006).
This flux of NO along with the presence of excessive ROS, may favor the reaction of NO with
superoxide anion producing peroxynitrite, resulting in increased oxidant stress and a net decrease
in NO production (Xia et al., 2006). Interestingly, this downregulation of NO production has also
been implicated in the pathogenesis of cardiovascular diseases, as decreased NO is associated with
impaired endothelium-dependent vasodilation (Liu et al., 2009).
NF-κB activation is also associated with the production of inflammatory cytokines. Inflammatory
cytokines play a role in endothelial cell injury by inducing the expression of cell adhesion molecules
(Lee et al., 2011b). Oxidative stress induction with H2O2 increased the production of pro-
inflammatory cytokines, including IL-2, IL-6, IL-8, IL-17, IFN-γ, and TNF-α in HUVEC. The addition
of astaxanthin to cell cultures had mixed effects on inflammatory cytokine production. Pre-
incubation with astaxanthin significantly decreased IL-8 and IFN-γ, but increased IL-17 production.
IL-8 has been implicated in the pathogenesis of a number of inflammatory diseases, as it is a chemo-
attractant and activator for neutrophils (Nyhlen et al., 2004). Additionally, IFN-γ is shown to
modulate IL-8 production in HUVEC (Nyhlen et al., 2004). In the present study, astaxanthin
appeared to have a protective effect against inflammation by downregulating IL-8 and IFN-γ
production.
IL-2, IL-6, IL-8, IFN-γ, and TNF-α are generally classified as Th1-modulating cytokines due to their
ability to induce immune responses, while IL-4 and IL-10 are generally classified as inhibitory or
Th2-modulatory cytokines due to their ability to inhibit pro-inflammatory cytokines. Th1-
modulating cytokines are known to block the proliferation of Th2 cells, leading to an inhibition of
Th2 effector function and a decrease in the production of anti-inflammatory cytokines. However,
stimulation with H2O2 in the present study also led to an increase in the production of the anti-
inflammatory cytokines IL-4 and IL-10; pretreatment with astaxanthin didn’t alter this production.
IL-4 and IL-10 have been shown to inhibt TNF-α and IL-6 production in isolated mononuclear cells,
Weslee Chew, Bridget D. Mathison, Lindsey L. Kimble, Philip F. Mixter, and Boon P. Chew / American Journal of
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12
while IL-4 stimulated IL-6 production in HUVEC (Guzdek et al., 2000). IL-4 and IL-10 also increased
IL-8 production in lipopolysaccharide-stimulated HUVEC (De Beaux et al., 1995). Tissue specific
differences in intracellular signaling pathways and complex interactions play critical roles in
cytokine production.
In conclusion, astaxanthin accumulates in HUVEC and incubation with this carotenoid protects
against inflammation and oxidative stress induced by H2O2. The action of astaxanthin in ROS
clearance helps restore cellular antioxidant function and inhibits ligands required for downstream
transcriptional activation and inflammatory gene expression. In general, the downregulation of NF-
κB and AP-1 resulted in the decreased production of inflammatory biomarkers involved in
endothelial dysfunction and CVD.
References
Bertram, J.S., Pung, A., Churley, M., Kappock, T.J., Wilkins, L.R., Cooney, R.V., 1991. Diverse carotenoids protect
against chemically induced neoplastic transformation. Carcinogenesis 12, 671-678.
http://dx.doi.org/10.1093/carcin/12.4.671
PMid:2013131
Cai, H., Li, Z., Davis, M., Kanner, W., Harrison, D., Dudley Jr., S., 2003. Akt-dependent phosphorylation of serine
1179 and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2
cooperatively mediate activation of the endothelial nitric-oxide synthase by hydrogen peroxide.
Molecular Pharmacology 63, 325-331.
http://dx.doi.org/10.1124/mol.63.2.325
PMid:12527803
Chen, X., Xu, J., Feng, Z., Fan, M., Han, J., Yang, Z., 2010. Simvastatin combined with nifedipine enhances
endothelial cell protection by inhibiting ROS generation and activating Akt phosphorylation. Acta
Pharmacologica Sinica 31, 813-820.
http://dx.doi.org/10.1038/aps.2010.58
PMid:20562903
Chew, B.P., Park, J.S., 2004. Carotenoid action on the immune response. Journal of Nutrition 134, 257-261.
Choi, Y., Jeong, Y., Lee, Y., Kwon, H., Kang, Y., 2005. (-)Epigallocatechin gallate and quercetin enhance survival
signaling in response to oxidant-induced human endothelial apoptosis. Journal of Nutrition 135, 707-713.
PMid:15795422
De Beaux, A., Maingay, J., Ross, J., Fearon, K., Carter, D., 1995. Interleukin-4 and interleukin- 10 increase
endotoxin-stimulated human umbilical vein endothelial cell interleukin-8 release. Journal of Interferon
and Cytokine Research 15, 441-445.
http://dx.doi.org/10.1089/jir.1995.15.441
PMid:7648446
Eligini, S., Arenaz, I., Barbieri, S.S., Faleri, M.L., Crisci, M., Tremoli, E., Colli, S., 2009. Cyclooxygenase-2
mediates hydrogen peroxide-induced wound repair in human endothelial cells. Free Radical Biology and
Medicine 46, 1428-1436.
http://dx.doi.org/10.1016/j.freeradbiomed.2009.02.026
PMid:19269318
Fassett, R.G., Coombes, J.S., 2011. Astaxanthin: A potential therapeutic agent in cardiovascular disease. Marine
Drugs 9, 447-465.
http://dx.doi.org/10.3390/md9030447
PMid:21556169 PMCid:3083660
Weslee Chew, Bridget D. Mathison, Lindsey L. Kimble, Philip F. Mixter, and Boon P. Chew / American Journal of
Advanced Food Science and Technology (2013) 1: 1-14
13
Foncea, R., Carvajal, C., Almarza, C., Leighton, F., 2000. Endothelial cell oxidative stress and signal
transduction. Biological Research 33, 89-96.
http://dx.doi.org/10.4067/S0716-97602000000200008
PMid:15693275
Guzdek, A., Stalinska, K., Guzik, K., Koj, A., 2000. Differential responses of hematopoietic and non-
hematopoietic cells to anti-inflammatory cytokines: IL-4, IL-13 and IL-10. Journal of Physiology and
Pharmacology 51, 387-399.
PMid:11016859
Hafizah, A.H., Zaiton, Z., Zulkhairi, A., Mohd Ilham, A., Nor Anita, M.M.N., Zaleha, A.M., 2010. Piper
sarmentosum as an antioxidant on oxidative stress in human umbilical vein endothelial cells induced by
hydrogen peroxide. Journal of Zhejiang University-Science (Biomedicine & Biotechnology) 11, 357-365.
Kao, C., Chen, L., Chang, Y., Yung, M., Hsu, C., Chen, Y., Lo, W., Chen, S., Ku, H., Hwang, S., 2010. Resveratrol
protects human endothelium from H2O2-induced oxidative stress and senescence via SirT1 activation.
Journal of Atherosclerosis and Thrombosis 17, 970-979.
http://dx.doi.org/10.5551/jat.4333
PMid:20644332
Kim, J., Na, H., Kim, C., Kim, J., Ha, K., Lee, H., Chung, H., Kwon, H.J., Kwon, Y., Kim, Y., 2008. The non-provitamin
a carotenoid, lutein, inhibits NF-κB-dependent gene expression through redox-based regulation of the
phosphatidylinositol 3-kinase/PTEN/Akt and NF-κB-inducing kinase pathways: Role of H2O2 in NF-κB
activation. Free Radical Biology and Medicine 45, 885-896.
http://dx.doi.org/10.1016/j.freeradbiomed.2008.06.019
PMid:18620044
Lee, S., Park, Y., Zuidema, M.Y., Hannink, M., Zhang, C., 2011a. Effects of interventions on oxidative stress and
inflammation of cardiovascular diseases. World Journal of Cardiology 3, 18-24.
http://dx.doi.org/10.4330/wjc.v3.i1.18
PMid:21286214 PMCid:3030733
Lee, S.J., Bai, S.K., Lee, K.S., Namkoong, S., Na, H.J., Ha, K.S., Han, J.A., Yim, S.V., Chang, K., Kwon, Y.G., Lee, S.K.,
Kim, Y.M., 2003. Astaxanthin inhibits nitric oxide production and inflammatory gene expression by
suppressing IκB kinase-dependent NF-κB activation. Molecules and Cells 16, 97-105.
PMid:14503852
Lee, S.M., Lee, Y.J., Kim, Y.C., Kim, J.S., Kang, D.G., Lee, H.S., 2011b. Vascular protective role of vitexicarpin
isolated from Vitex rotundifolia in human umbilical vein endothelial cells. Inflammation 35, 584-593.
http://dx.doi.org/10.1007/s10753-011-9349-x
PMid:21614554
Liu, H., Li, W., Xu, G., Li, X., Bai, X., Wei, P., Yu, C., Du, Y., 2009. Chitosan oligosaccharides attenuate hydrogen
peroxide-induced stress injury in human umbilical vein endothelial cells. Pharmacological Research 59,
167-175.
http://dx.doi.org/10.1016/j.phrs.2008.12.001
PMid:19121394
Lin, Y., Zhen, Y., Wei, J., Liu, B., Yu, Z., Hu, G., 2011. Effects of Rhein lysinate on H2O2-induced cellular
senescence of human umbilical vascular endothelial cells. Acta Pharmacologica Sinica 32, 1246-1252.
http://dx.doi.org/10.1038/aps.2011.101
PMid:21909125
Lloyd-Jones, D., Adams, R.J., Brown, T.M., Carnethon, M., Dai, S., De Simone, G., Ferguson, T.B., Ford, E., Furie,
K., Gillespie, C., Go, A., Greenlund, K., Haase, N., Hailpern, S., Ho, P.M., Howard, V., Kissela, B., Kittner, S.,
Lackland, D., Lisabeth, L., Marelli, A., McDermott, M.M., Meigs, J., Mozaffarian, D., Mussolino, M., Nichol, G.,
Roger, V.L., Rosamond, W., Sacco, R., Sorlie, P., Stafford, R., Thom, T., Wasserthiel-Smoller, S., Wong, N.D.,
Wylie-Rosett, J., 2010. Executive summary: Heart disease and stroke statistics--2010 update: A report
from the American Heart Association. Circulation 121, 948-954.
http://dx.doi.org/10.1161/CIRCULATIONAHA.109.192666
PMid:20177011
Weslee Chew, Bridget D. Mathison, Lindsey L. Kimble, Philip F. Mixter, and Boon P. Chew / American Journal of
Advanced Food Science and Technology (2013) 1: 1-14
14
Lo, Y.Y.C., Wong, J.M.S., Cruz, T.F., 1996. Reactive oxygen species mediate cytokine activation of c-Jun NH2-
terminal kinases. Journal of Biological Chemistry 271, 15703-15707.
http://dx.doi.org/10.1074/jbc.271.26.15703
PMid:8663189
Mo, S., Son, E., Lee, S., Lee, S., Shin, D., Pyo, S., 2007. CML-1 inhibits TNF-α-induced NF-κB activation and
adhesion molecule expression in endothelial cells through inhibition of IκBα kinase. Journal of
Ethnopharmacology 109, 78-86.
http://dx.doi.org/10.1016/j.jep.2006.07.006
PMid:16920299
Moutet, M., d'Alessio, P., Malette, P., Devaux, V.e., Chaudi`ere, J., 1998. Glutathione peroxidase mimics prevent
TNF-α- and neutrophil-induced endothelial alterations. Free Radical Biology and Medicine 25, 270-281.
http://dx.doi.org/10.1016/S0891-5849(98)00038-0
Naguib, Y.M., 2000. Antioxidant activities of astaxanthin and related carotenoids. Journal of Agricultural and
Food Chemistry 48, 1150-1154.
http://dx.doi.org/10.1021/jf991106k
PMid:10775364
Nyhlen, K., Gautam, C., Andersson, R., Srinivas, U., 2004. Modulation of cytokine-induced production of IL-8 in
vitro by interferons and glucocorticosteroids. Inflammation 28, 77-88.
http://dx.doi.org/10.1023/B:IFLA.0000033023.76110.51
PMid:15379213
Olszanecki, R., Gebska, A., Korbut, R., 2006. Production of prostacyclin and prostaglandin E2 in resting and IL-
-stimulated A549, HUVEC and hybrid EA.HY 926 cells. Journal of Physiology and Pharmacology 57,
649-660.
PMid:17229988
Park, J., Kim, H., Mathison, B., Hayek, M., Massimino, S., Reinhart, G., Chew, B., 2010. Astaxanthin uptake in
domestic dogs and cats. Nutrition and Metabolism 7, 52-59.
http://dx.doi.org/10.1186/1743-7075-7-52
PMid:20565958 PMCid:2898833
Pashkow, F., Watumull, D., Campbell, C., 2008. Astaxanthin: A novel potential treatment for oxidative stress
and inflammation in cardiovascular disease. The American Journal of Cardiology 101 (Suppl), 58-68.
http://dx.doi.org/10.1016/j.amjcard.2008.02.010
PMid:18474276
Spanier, G., Xu, H., Xia, N., Tobias, S., Deng, S., Wojnowski, L., Forstermann, U., Li, H., 2009. Resveratrol reduces
endothelial oxidative stress by modulating the gene expression of superoxide dismutase 1 (SOD1),
glutathione peroxidase 1 (GPx1) and NADPH oxidase subunit (NOX4). Journal of Physiology and
Pharmacology 60, 111-116.
PMid:20083859
Xia, Z., Liu, M., Wu, Y., Sharma, V., Luo, T., Ouyang, J., McNeill, J., 2006. N-acetylcysteine attenuates TNF-α
induced human vascular endothelial cell apoptosis and restores eNOS expression. European Journal of
Pharmacology 550, 134-142.
http://dx.doi.org/10.1016/j.ejphar.2006.08.044
PMid:17026986
... Anticancer [92] Antiproliferative activity [93] Bone disease [94] Immunoregulatory activity [95] Antimicrobial activity [92,96,97] Cytotoxic activity [98] Antityrosinase activity [98] Neuroprotection [99][100][101] Immune response [82,102] Cardiovascular prevention [15,[103][104][105] Anti-diabetes [50,106,107] Anti-hepatoprotective [108] Anti-gastric activity [109,110] Anti-inflammatory [105,[111][112][113][114] Anti-skin cancer [115][116][117] Protection from UV rays [117] Antioxidant activity [11,13,18,69,95,97,98,[117][118][119][120][121][122][123] process [40]. It demonstrates the important role of a controlled ROS generation in the proper functioning of the sperm, thus providing ROS with the main role in the process of maturation, other than the detrimental factor previously evaluated by the sperm [10]. ...
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... For this reason, very few studies have been conducted to study its effects. However, Chew et al. (2013), were able to discover that astaxanthin slowly permeates into cells, peaking mostly between 24 and 48 h. In their study, hydrogen peroxide was used to induce oxidative damage and astaxanthin was used as an antioxidative treatment. ...
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Silencing information regulator (SirT1), a NAD-dependent histone deacetylase, is an essential mediator of longevity in normal cells by calorie restriction. SirT1 has many biological functions, including transcription regulation, cell differentiation inhibition, cell cycle regulation, and anti-apoptosis. Resveratrol (RV)-induced SirT1 activation also improves endothelial dysfunction and suppresses vascular inflammation. In this study, we investigated the roles of RV-induced SirT1 activation in endothelial cells under oxidative stress. SirT1 mRNA expression levels were examined in the endothelium layer (endothelial cells) of cardiac coronary vessels from patients receiving coronary artery bypass graft surgery (CABG) surgery and aged rats using reverse transcriptase polymerase chain reaction (RT-PCR). To further explore the effect of SirT1 activation on oxidative stress-induced aging, senescence-associated β-galactosidase (SA-β-gal) expression in RV-treated human umbilical vein endothelial cells (HUVECs) with or without H(2)O(2) treatment was evaluated. SirT1 expression was decreased in aged and atherosclerotic vessels in vivo, and significantly reduced in endothelial cells purified from vessel tissues. Furthermore, SirT1 levels were dose-dependently increased in RV-treated HUVECs. The SA-β gal assay showed that RV inhibited the senescent phenotype of H(2)O(2)-treated HUVECs. Reactive oxygen species (ROS) production and the percentage of cells positive for SA-β gal were significantly increased in siRNA-SirT1 (knockdown of SirT1 expression)-treated HUVEC cells. Importantly, the treatment effect of RV was significantly abolished in the oxidative effects of H(2)O(2)-treated HUVECs by siRNA-SirT1. Our data suggested that SirT1 could be a crucial factor involved in the endothelial cells of atherosclerotic CAGB patients and aging rats. RV is a potential candidate for preventing oxidative stress-induced aging in endothelial cells. RV may also prevent ROS-induced damage via increased endothelial SirT1 expression.
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