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© 1997 American Heart Association, Inc.
Articles |
Genistein, the Dietary-Derived Angiogenesis Inhibitor, Prevents LDL Oxidation and Protects Endothelial Cells From Damage by Atherogenic LDL
From the Clinical Institute of Medical and Chemical Laboratory Diagnostics (S.K.), the Institute of Molecular Genetics (M.H.), the Institute of Tumor Biology and Cancer Research (C.S.), the Clinic for Internal Medicine II, Department of Angiology (H.E.), and the Institute of Medical Chemistry (I.H, B.G.), University of Vienna, Vienna, Austria.
Correspondence to Bernhard Gmeiner, Institute of Medical Chemistry, University of Vienna, Währingerstr 10, A-1090 Vienna, Austria. E-mail stylianos.kapiotis{at}univie.ac.at
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Abstract |
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 Top Abstract IntroductionMethodsResults and DiscussionReferences |
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Abstract There is now growing evidence that the oxidative modification of LDL plays a potential role in atherosclerosis. In this study, genistein, a compound derived from a soy diet with a flavonoid chemical structure (4',5,7-trihydroxyisoflavone), which was found to inhibit angiogenesis, has been evaluated for its ability to act as an LDL antioxidant and a vascular cell protective agent against oxidized LDL. The results showed that genistein was able to inhibit the oxidation of LDL in the presence of copper ions or superoxide/nitric oxide radicals as measured by thiobarbituric acid-reactive substance formation, alteration in electrophoretic mobility, and lipid hydroperoxides. Bovine aortic endothelial cell- and human endothelial cell-mediated LDL oxidation was also inhibited in the presence of genistein. The 7-O-glucoside of genistein, genistin, was much less effective in inhibiting LDL oxidation in the cell-free and cell-mediated lipoprotein-oxidating systems. Incubating human endothelial cells in the absence or presence of genistein and challenging the cells with already oxidized lipoprotein revealed that in addition to its antioxidative potential during LDL oxidating processes, genistein effectively protected the vascular cells from damage by oxidized lipoproteins. The tyrosine kinase inhibitor genistein was found to block upregulation of two tyrosine-phosphorylated proteins of 132 and 69 kDa in endothelial cells induced by oxidized LDL. Parallel experiments with the inactive analogue daidzein, however, showed that the cytoprotective effect of the isoflavones seems not to be dependent on tyrosine phosphorylation. Our findings will support the suggested and documented beneficial action of a soy diet in preventing chronic vascular diseases and early atherogenic events.
Key Words: oxidized LDL • genistein • vasoprotection • flavonoids • atherosclerosis
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Introduction |
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TopAbstractIntroductionMethodsResults and DiscussionReferences |
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The onset of atherosclerosis is a complex process, but there is now some evidence that the oxidative modification of LDL may play a key role in early atherogenic events.1–4 Minimally oxidized LDL activates endothelial cells to attract and bind monocytes, and consecutively foam cells are formed, leading to the appearance of the fatty streak lesion.5,6
LDL oxidation can be mediated by various systems such as transition metal ions (copper and iron), myeloperoxidases, nitric oxide/superoxide radicals, hypochlorite, and vascular cells (endothelial cells, macrophages, and smooth muscle cells).4,7–11
Hence, these observations have led to in vitro and in vivo studies focusing on the prevention of LDL oxidation. The antioxidant probucol has been found to inhibit the formation of foam cells, and the radical scavenger butylated hydroxytoluene when administered to rats was able to suppress the onset of atherosclerotic lesions.12–14
From a chemical point of view, the phenols and polyphenolic compounds are the most promising synthetic or naturally occurring compounds (flavonoids) with respect to inhibition of atherogenic (oxidative) modifications of LDL. Thus, a variety of these substances have been tested for their antioxidative potential.15–18
Recently, genistein, a dietary-derived isoflavonoid, has been found to be a potent inhibitor of endothelial cell proliferation and angiogenesis.19 Owing to the relatively high excretion of genistein found in urine of vegetarians (about 6 µmol/24 h in contrast with the concentration in urine of omnivores (0.18 µmol/24 h), these authors speculated that genistein might contribute to the known preventive effect of a plant-based diet on chronic (neovascular) diseases.19
With respect to its polyphenolic chemical structure (Fig 1
), we have tested this compound for
its ability to inhibit LDL oxidation, a property that might have
implications in preventing the risk of coronary heart disease.
Genistein inhibited the oxidation process of LDL as well in a cell-free
as in an endothelial cell model system. In addition to
its antioxidant potential, the compound was also able to act as a
vascular protective agent, that is, genistein prevented the
endothelial cells from the cytotoxic effect of already
oxidized LDL.
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Methods |
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TopAbstractIntroductionMethodsResults and DiscussionReferences |
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Genistein (4',5,7-trihydroxyisoflavone), genistin (4',5,7-trihydroxyisoflavone-7-glucoside), SIN-1, 1,1,3,3,-tetrameth-oxypropane, and 2-thiobarbituric acid were from Sigma Chemical Co. Daidzein (4',7-trihydroxyisoflavone) was from Calbiochem. The phosphotyrosine antibody was purchased from Upstate Biotechnology, Inc. All other chemicals used were of analytical grade.
LDL Isolation
LDL was isolated by ultracentrifugation as
reported previously.20 The final preparation was
dialyzed against 150 mmol/L NaCl containing 0.1
mmol/L EDTA and filter-sterilized. Protein concentration was
estimated by a commercial test kit (Bio-Rad Laboratories) using bovine
serum albumin as a standard.
Endothelial Cell Cultures
BAECs were prepared from fresh (within 30 minutes of death),
removed aortas: the aorta was flushed with sterile PBS, tied off at one
end, and cannulated at the other with a 50-mL syringe containing
sterile PBS, which was infused to the lumen before the vessel was
transported to the laboratory within 1 hour. The
endothelial cells were then isolated by enzymatic
digestion. After the intercostal arteries were ligated, the aorta was
washed with sterile PBS, and 10 mL of collagenase (type Ia,
Sigma Chemical Co; final concentration 200 U/ml) in PBS for 15 minutes
at 37°C. The collagenase solution was collected in a
sterile tube, and remaining cells were harvested with 10 mL of PBS.
Cells were spun down for 10 minutes at 1500 rpm at room temperature.
Supernatant was discarded, and the cells were resuspended in RPMI-1640
medium supplemented with 20% fetal calf serum in T200 culture flasks.
The culture medium was replaced every 2 days. For experiments, cells
were passaged into 35-mm culture dishes, where they reached confluency
within 2 to 3 days. Cell numbers were
6.4±0.3x105 cells/35-mm well (n=10). Passages
10 to 20 were used for experiments. HUVECs were prepared and maintained
in culture as previously reported.21
LDL Oxidation
Cell-Free Oxidation System
Before oxidation experiments, LDL was applied to a PD-10 column
(Pharmacia BioTech) equilibrated with 150 mmol/L NaCl to
get rid of EDTA.
LDL was oxidized by incubating LDL (50 to 200 µg/mL) either in the presence of copper ions (10 µmol/L) or SIN-1 (1 mmol/L) at 37°C in 150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.4 (NaCl/Tris-buffer), for the indicated time. The oxidation reaction was stopped by the addition of EDTA (100 µmol/L). For some cell culture experiments LDL was oxidized with copper ions in RPMI-1640 medium (PAA) as indicated in the figure legends.
Cell-Mediated Oxidation System
When cell-mediated LDL oxidation was performed, BAEC or HUVEC
confluent monolayers were washed with RPMI-1640 medium and subsequently
incubated with LDL (50 µg/mL) in RPMI-1640 medium supplemented
with 2.5 µmol/L Cu2+ 11 for 24
hours. Results were obtained with two different passages of BAECs and
four different HUVEC preparations. Representative
experiments are depicted.
Analysis of LDL Oxidation
Thiobarbituric Acid Assay
LDL oxidation products were assayed as TBARS as
described.20 Concentrations of TBARS were
calculated using malondialdehyde generated from
1,1,3,3,-tetramethoxypropane as a standard.
Lipid Hydroperoxide Assay
Total lipid hydroperoxides were assayed according to el Saadani
et al22 as modified by Wallin and
Camejo.23 The concentration of hydroperoxides was
calculated from the molar absorption coefficient of
2.46x104
mol/L·cm-1 at 365 nm.
Lipid Electrophoresis
Aliquots (10 µL) of treated or untreated LDL (200
µg/mL) were applied to agarose gels (1% in veronal buffer)
and run for 90 minutes. Lipoproteins were detected by precipitation
with phosphotungstic acid/MgCl2 according to the
supplier of the analytical system (Lipidophor All In, Immuno AG).
Measurement of relative electrophoretic mobility was taken as an
indicator of LDL oxidation,24 setting the
electrophoretic mobility of native (untreated) LDL arbitrarily as
1.
Cytotoxicity Assay
LDH activity in the cell culture supernatants was measured as an
indicator of cell cytotoxicity. LDH was estimated by a commercially
available spectrophotometric assay on a Hitachi 717 analyzer
(Boehringer Mannheim).
Western Blot Analysis of Tyrosine Phosporylated
Proteins
Confluent HUVEC monolayers were washed twice with RPMI-1640
medium without fetal calf serum and were exposed to LDL (200
µg/mL) and Cu2+ (2.5
µmol/L) with and without genistein (100 µmol/L)
or daidzein (100 µmol/L) for 24 hours. Thereafter,
supernatant was removed, cells were washed twice with PBS, and 200 µL
of sample buffer containing 2% SDS, 10% glycerol, 0.125 mol/L
Tris-HCl (pH 6.8), 0.00125% bromphenol blue, and 2% 2-mercaptoethanol
was added to monolayers. The lysate was boiled for 3 minutes. and
proteins were separated by 7.5% SDS-PAGE. After electrophoresis,
proteins were transferred to nitrocellulose membranes and
tyrosine-phosphorylated proteins were detected with a
mouse monoclonal anti-phosphotyrosine antibody (1 µg/mL) and
visualized by enhanced chemoluminiscence using a commercially available
kit (Amersham). Prestained molecular weight markers ranging from
47 000 to 205 000 (Bio-Rad) were used as standards for the
SDS-PAGE.
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Results and Discussion |
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TopAbstractIntroductionMethodsResults and DiscussionReferences |
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LDL Oxidation in Cell Free Systems
Due to its polyphenolic structure we have tested genistein for its ability to inhibit LDL oxidation. As can be seen in Fig 2
, when genistein was added to the LDL
oxidation system (50 µg/mL of LDL, 10 µmol/L of
Cu2+), a strong inhibition of TBARS formation was
observed. In contrast, genistin, the 7-O-glucoside of
genistein, was less active in inhibiting LDL oxidation. This may be due
to the loss of the polyphenolic character by the O-glucoside
formation or to the more hydrophilic property of genistin, making
access of the compound to the lipid environment, the target of
oxidative damage, more difficult. To test this, the genistein analogue
daidzein, which is more similar to genistein in its lipophilic
properties than genistin, was evaluated. Daidzein showed a somewhat
stronger effect than genistin, but did not reach the
inhibitory capacity of genistein. This points to a role of
the polyphenolic molecule structure rather than the lipophilicity in
the antioxidant properties of these compounds.
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, genistin 
, and
daidzein 
on LDL oxidation. LDL (50 µg/mL) was incubated in
NaCl/Tris buffer with 10 µmol/L Cu2+ in the absence
or presence of genistein, genistin, or daidzein for 4 hours at 37°C.
LDL oxidation was assessed by the measurement of TBARS.
Measurement of total lipid hydroperoxides as an additional
parameter of lipid oxidation revealed the same results as
found for TBARS formation. In the presence of genistein, less
hydroperoxides were formed during the process of
Cu2+-promoted LDL oxidation. (Fig 3).
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The oxidation of LDL causes an alteration in the electrophoretic
mobility of the lipoprotein due to an increase in net charge of the
protein.24 Hence, the estimation of the relative
electrophoretic mobility is a further measure of LDL oxidative
modification. Therefore, LDL was subjected to oxidation in the absence
or presence of genistein (100 µmol/L), and the relative
electrophoretic mobility was estimated. As can be seen in Fig 4, genistein was able to prevent the
time-dependent increase in REM when present during LDL
oxidation.
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or presence
NO radicals (NO·) and superoxide/nitric oxide
(O2
-/NO·) radicals have been
shown to initiate LDL oxidation.9,10 Because these radical
species are formed in vivo, it was suggested that they may be able to
contribute to LDL oxidation in the vascular
system.9,28 The sydnonimine SIN-1 had been shown
to generate O2-/NO· radicals
synergistically in solution, and it had been reported that isolated
LDLs or membrane lipids were oxidized in the presence of
SIN-1.10,29 We have therefore tested the
influence of genistein on this particular kind of LDL oxidative
modification. LDL (200 µg/mL) was incubated for 16 hours at
37°C with or without SIN-1 (1 mmol/L) in the absence or
presence of genistein. As depicted in Fig 5, genistein, when present during
O2-/NO·-initiated LDL oxidation, was
able to inhibit the radical driven reaction. At 50 µmol/L
of genistein there were about 50% less lipid hydroperoxides formed
compared with controls without genistein. Thus, genistein was able to
overcome the LDL oxidation in two alternative LDL-oxidizing systems,
viz, metal ion-dependent and metal ion-independent
(O2·-/NO·) oxidation. Both
systems have been suggested to work in vivo, making LDL
atherogenic.10,28,30
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LDL Oxidation by Endothelial Cells
A variety of cells have been shown to be able to oxidize
LDL.3 The modified lipoprotein can be taken up by
the cells, leading to several changes in cellular behavior, and
finally, when taken up in excess oxidized LDL can cause cytotoxic
effects.3,6
Thus, we have tested the influence of genistein (genistin) on LDL
oxidation by endothelial cells. When tested in a bovine
(BAEC) and human (HUVEC) endothelial cell system,
genistein was able to overcome the LDL-oxidizing effect of the cells.
At 100 µmol/L genistein, there was about 50% inhibition
of cell-mediated lipoprotein oxidation observed as indicated by TBARS
formation in the culture media (Fig 6).
As found in the cell-free oxidation system (see Fig 2
), genistin was
less effective or even ineffective in preventing LDL oxidation (Fig 6)
by both cell types.
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As can be seen in Fig 7A, parallel
estimation of LDH activity taken as an indicator of cytotoxic effects
revealed that as genistein inhibited LDL oxidation in the HUVEC
cultures, the cytotoxic effect was also inhibited. In cultures (50
µg/mL of LDL) where maximal LDL oxidation occurred, 80 U/L of
LDH was measured in contrast with 28 U/L in controls (no LDL added).
Genistein, but not genistin, inhibited LDH release in a
concentration-dependent manner. At 50 to 100 µmol/L
genistein, the enzyme activity reached control values. In addition,
microscopic examination of the cells showed that genistein, in contrast
with genistin, prevented the typical morphologic alterations (rounding)
of the cells due to the cytotoxic effect of oxidized LDL (Fig 7B). The
genistein analogue daidzein was as effective as genistein in its
endothelial cell protective activity as assessed by LDH
release and microscopy (data not shown). None of the flavonoids tested
had any effect on basal LDH release from endothelial
cells.
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To assess whether genistein has to be present during LDL oxidation
or genistein pretreatment could also decrease the ability of
endothelial cells to oxidize LDL, HUVEC cultures were
preincubated in the absence or presence of 100 µmol/L
genistein for 24 hours. After the cell were washed, medium containing
50 µg/mL LDL was added, and the time-dependent lipoprotein
oxidation was monitored. As depicted in Fig 8, genistein-pretreated cells showed very
similar kinetics with respect to LDL oxidation compared with controls.
In parallel cultures supplemented with 100 µmol/L
genistein, LDL oxidation was inhibited. Thus, pretreatment of the cells
with genistein did not prevent the cell-mediated atherogenic
modification of LDL under the conditions applied.
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, pregenistein. In cultures designated co-genistein
The above results showed that genistein was able to inhibit LDL
oxidation in cell-free and cell-mediated lipoprotein-oxidating systems.
Next we addressed the question whether genistein could protect
endothelial cells from the damaging effect of oxidized
LDL. Therefore, LDL (50 µg/mL) was preoxidized in RPMI-1640
for 24 hours at 37°C with 10 µmol/L
Cu2+ and subsequently HUVEC cultures were treated
with already oxidized LDL (50 µg/mL) in the absence or
presence of genistein (100 µmol/L). Cytotoxicity was
monitored by LDH release (Fig 9A) and
microscopic examination of the cultures (Fig 9B). As seen in Fig 9, genistein was able to protect endothelial cells from
the deleterious effect of oxidized LDL. It must be noted that
estimation of TBARS in the culture media at the end of incubation
revealed that this effect was not due to any genistein-mediated
inhibition of further LDL oxidation, which may occur by the cells, as
TBARS concentrations were virtually equal in culture media in the
absence or presence of genistein (1.2 µmol/L versus
1.3 µmol/L, respectively). In addition, following the
kinetics (0 to 48 hours) of LDL preoxidation under the above conditions
(RPMI-1640, 37°C, 10 µmol/L Cu2+)
the results showed that maximal oxidation of the lipoprotein had
occurred after 24 hours (data not shown). Thus, "maximally"
oxidized LDL had been added to the cultures.
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Therefore, one can assume that genistein could act as an efficacious vascular protective agent, that is, could protect endothelial cells from the damaging effect of atherogenic LDL. Thus, in this article, in addition to an antioxidative activity, a vascular protective property of a flavonoid is described. However, the possibility that the vascular protective effect of genistein is due to its antioxidant capacity cannot be excluded.
Genistein is known as a tyrosine kinase inhibitor and has
been shown to counteract cellular responses to a variety of
compounds.31–36 In this respect, it is of
interest to note that a preliminary study showed that oxidized LDL
stimulated the tyrosine phosphorylation in rabbit
aortic endothelial cells.37,38 To
explore whether genistein exerts its protective action against oxidized
LDL via an inhibition of tyrosine kinase-dependent pathways, HUVECs in
the absence or presence of oxidized LDL and genistein were
analyzed with respect to
tyrosine-phosphorylated proteins. As shown in Fig 10, in untreated cultures two
tyrosine-phosphorylated proteins with molecular weights
of 132 000 and 69 000 were detected by use of a phosphotyrosine
antibody. Cell-mediated LDL oxidation stimulated tyrosine
phosphorylation of both proteins. Genistein (100
µmol/L) completely inhibited the effect of oxidated LDL.
Daidzein, commonly used as an inactive analogue of genistein with
respect to inhibition of tyrosine kinases,31 was
not able to inhibit increased tyrosine phosphorylation
due to oxidated LDL. Taking into account that both genistein and
daidzein are cytoprotective against oxidized LDL, one could speculate
that the effect of genistein (daidzein) described in this study may not
be due to inhibition of oxidized LDL-stimulated tyrosine
phosphorylation in HUVECs.
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Recently, Kanazawa et al39 reported on the influence of soy protein diet on the peroxidizability of lipoproteins in cerebrovascular diseases. These authors showed that the administration of soycreme to patients suppressed the oxidizability of lipoproteins promoted by copper ions. Taking our results into account, it is intriguing to speculate that this could be due to the action of genistein, the soy diet-derived antioxidative compound. However, to verify this suggestion, further investigations would be necessary.
In any case, our results showed that genistein is a potent antioxidant for in vitro LDL oxidation and in addition is an endothelial cell protective agent against the cytotoxic effect of oxidized LDL. Hence, our findings will support the suggested and documented beneficial action of soy diet in preventing chronic vascular diseases40,41 and early atherogenic events.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by the Wissenschaftlicher Fonds des Bürgermeisters der Bundeshauptstadt Wien.
Received March 18, 1997; accepted August 25, 1997.
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