Relationship between protective effect of xanthone on endothelial cells and endogenous nitric oxide synthase inhibitors

Article abstract of DOI:10.1016/j.bmc.2003.08.015

1,3,5,6-tetrahydroxyxanthone was synthesized. The relationship between protective effect of xanthone on endothelial cells and endogenous nitric oxide synthase inhibitors was investigated. Endothelial cells were treated with ox-LDL (100 μg/mL) for 48 h. Ad

Full text of DOI:10.1016/j.bmc.2003.08.015

Bioorganic & Medicinal Chemistry 11 (2003) 5171–5177
Relationship between Protective Effect of Xanthone on
Endothelial Cells and Endogenous Nitric Oxide Synthase
Inhibitors
a
b
a
b
a
De-Jian Jiang, Gao-Yun Hu, Jun-Lin Jiang, Hong-Lin Xiang, Han-Wu Deng
a,
and Yuan-Jian Li *
aDepartment of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha 410078, China
bDepartment of Medicinal Chemistry, School of Pharmaceutical Sciences, Central South University, Changsha 410078, China
Received 15 May 2003; accepted 15 August 2003
Abstract—1,3,5,6-tetrahydroxyxanthone was synthesized. The relationshipbetween pr otective effect of xanthone on endothelial
cells and endogenous nitric oxide synthase inhibitors was investigated. Endothelial cells were treated with ox-LDL (100 mg/mL) for
4
8 h. Adhesion of monocytes to endothelial cells and release of lactate dehydrogenase (LDH) was determined. Levels of tumor
necrosis factor-a (TNF-a), monocyte chemoattractant protein-1 (MCP-1), nitric oxide (NO) and asymmetric dimethylarginine
ADMA, an endogenous inhibitor of nitric oxide synthase) in conditioned medium and activity of dimethylarginine dimethylamino-
(
hydrolase (DDAH) in endothelial cells were measured. Incubation of endothelial cells with ox-LDL (100 mg/mL) for 48 h markedly
enhanced the adhesion of monocytes to endothelial cells, increased the release of LDH, the levels of TNF-a, MCP-1 and ADMA,
and decreased the content of NO and the activity of DDAH. Xanthone (1,3,5,6-tetrahydroxyxanthone) (1, 3 or 10 mmol/L) sig-
nificantly inhibited the increased adhesion of monocytes to endothelial cells and attenuated the increased levels of LDH, MCP-1
and ADMA induced by ox-LDL. Xanthone (1,3,5,6-tetrahydroxyxanthone) (3 or 10 mmol/L) significantly attenuated the increased
level of TNF-a and decreased level of NO and activity of DDAH by ox-LDL. The present results suggest that xanthone (1,3,5,6-
tetrahydroxyxanthone) preserves endothelial cells and inhibits the increased adhesion of monocytes to endothelial cells induced by
ox-LDL, and that the protective effect of xanthone (1,3,5,6-tetrahydroxyxanthone) on endothelial cells is related to reduction of
ADMA concentration via increase of DDAH activity.
#
2003 Elsevier Ltd. All rights reserved.
Introduction
xanthone compounds, extracted from Swertia davidi
Franch (Gentianaceae), protect the myocardium against
ischemia-reperfusion injury and attenuate the inhibition
of vascular endotheliun-dependent relaxation induced
Xanthones, a kind of ployphenolic compounds that
commonly occur in plants, have widely been synthe-
sized. A great deal of information has been demon-
strated that xanthones and xanthone derivatives have
extensive pharmacological activities such as antioxida-
tion, antihypertension and inhibition of platelet
5ꢀ7
by lysophosphatidylcholine (LPC).
There is growing evidence that endothelial dysfunction
and later adhesion of circulating monocytes to endo-
thelium play an important role in early events of ather-
1
ꢀ3
aggregation.
Recently, it was reported that some
8
synthetic xanthones show the strong property of anti-
inflammation, which significantly inhibited the
increased expression of intercellular adhesion molecule-
(ICAM-1) induced by tumor necrosis factor-a (TNF-
a), an inflammatory cytokine, in cultured human endo-
thelial cells. Our recent work has shown that some
ogenesis. It is known that endothelium-derived nitric
oxide (NO), which regulates endothelial function, is
synthesized from l-arginine by NO synthase (NOS) in
endothelial cells. Recently, it has been found that endo-
genous inhibitors of NOS such as asymmetric dimethy-
larginine (ADMA), which inhibits NO synthesis, are
significantly increased in animals and patients with
atherosclerosis, and ADMA has been thought as a key
1
4
9ꢀ12
factor contributing endothelial dysfuction. It has been
reported that oxidized low-density lipoprotein (ox-LDL)
*
0
Correspoinding author. Tel.: +86-731-480-5441; fax: +86-731-265-
442; e-mail: yuan_jianli@yahoo.com
0
968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.08.015

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D.-J. Jiang et al. / Bioorg. Med. Chem. 11 (2003) 5171–5177
or TNF-a increases the level of ADMA in cultured
L). However, Xanthone or probucol itself had no effect
on the activity of LDH (Fig. 1).
1
3
human endothelial cells. Since some inflammatory
cytokines induces production of ADMA and some
xanthones have anti-inflammation properties, we exam-
ined the relationshipbetween the pr otection of xan-
thone (1,3,5,6-tetrahydroxyxanthone) of endothelial
cells and reduction of ADMA in cultured endothelial
cells treated with ox-LDL.
Adhesion of the monocytes to endothelial cells
Treatment with ox-LDL (100 mg/mL) for 48 h caused a
significant increase in the adhesion of monocytes to
endothelial cells. Xanthone (1, 3 or 10 mmol/L) sig-
nificantly attenuated the increase in adhesion of mono-
cytes by ox-LDL. The increased adhesion of monocytes
by ox-LDL was also attenuated by treatment with pro-
bucol (5 mmol/L). Xanthone or probucol itself had no
effect on the adhesion of monocytes to endothelial cells
Results
Activity of lactate dehydrogenase (LDH)
(
Fig. 2).
Treatment with ox-LDL (100 mg/mL) for 48 h sig-
nificantly increased the activity of LDH in the condi-
tioned medium. Xanthone (1, 3 or 10 mmol/L)
significantly inhibited the elevated activity of LDH by
ox-LDL. The increased activity of LDH by ox-LDL
was also inhibited by treatment with probucol (5 mmol/
Concentrations of TNF-ꢀ
Treatment with ox-LDL (100 mg/mL) for 48 h caused a
significant increase in concentrations of TNF-a. Xan-
thone (3 or 10 mmol/L) significantly inhibited the ele-
vated concentration of TNF-a by ox-LDL (100 mg/mL).
Probucol (5 mmol/L) also markedly inhibited the ele-
vated concentration of TNF-a by ox-LDL. However,
Xanthone or probucol itself had no effect on the
concentration of TNF-a (Fig. 3).
Concentrations of monocyte chemoattractant protein-1
(MCP-1)
Treatment with ox-LDL (100 mg/mL) for 48 h caused a
significant decrease in concentrations of MCP-1 in the
medium. Xanthone (1, 3 or 10 mmol/L) significantly
attenuated the increased level of MCP-1 by ox-LDL.
The increased level of MCP-1 by ox-LDL was also
attenuated by treatment with probucol (5 mmol/L).
Xanthone or probucol itself had no effect on the
concentration of MCP-1 (Fig. 4).
Figure 1. Effect of xanthone on activity of LDH in the conditioned
medium. Xan: 1,3,5,6-tetrahydroxyxanthone. MeansꢁSEM, n=6.
Concentrations of nitrite/nitrate
++
p<0.01 versus control, *p<0.05, **p<0.01 versus ox-LDL.
Treatment with ox-LDL (100 mg/mL) for 48 h caused a
significant decrease in concentrations of nitrite/nitrate
Figure 2. Effect of xanthone on adhesion of moncytes to endothelial
cells. Xan: 1,3,5,6-tetrahydroxyxanthone. MeansꢁSEM, n=6.
Figure 3. Effect of xanthone on levels of TNF-a in the conditioned
medium. Xan: 1,3,5,6-tetrahydroxyxanthone. MeansꢁSEM., n=6.
++
++
p<0.01 versus control, *p<0.05, **p<0.01 versus ox-LDL.
p<0.01 versus control, *p<0.05, **p<0.01 versus ox-LDL.

D.-J. Jiang et al. / Bioorg. Med. Chem. 11 (2003) 5171–5177
5173
in the medium. Xanthone (3 or 10 mmol/L) significantly
attenuated the decreased level of nitrite/nitrate by ox-
LDL. The decreased level of nitrite/nitrate by ox-LDL
was also attenuated by treatment with probucol (5
mmol/L). Xanthone or probucol itself had no effect on
the concentration of nitrite/nitrate (Fig. 5).
Activity of dimethylarginine dimethylaminohydrolase
(DDAH)
DDAH activity was significantly decreased in the endo-
thelial cells treated with ox-LDL (100 mg/mL) for 48 h
(52.0ꢁ7.5% vs control, p<0.01). Xanthone (3 or 10
mmol/L) significantly attenuated inhibition of endothe-
lial DDAH activity by ox-LDL. Probucol (5 mmol/L)
also markedly attenuated inhibition of endothelial
DDAH activity by ox-LDL. However, Xanthone or
probucol itself had no effect on the activity of DDAH
(Fig. 7).
Concentrations of ADMA
The basal level of ADMA in the medium was
0
.45ꢁ0.07 mmol/l. Treatment with ox-LDL (100 mg/
mL) for 48 h significantly increased concentrations of
ADMA (1.35ꢁ0.13 mmol/l, p<0.01 vs control). Xan-
thone (1, 3 or 10 mmol/L) significantly inhibited the ele-
vated concentration of ADMA by ox-LDL. Probucol (5
mmol/L) also markedly inhibited the elevated concen-
tration of ADMA by ox-LDL (100 mg/mL). However,
Xanthone or probucol itself had no effect on the con-
centration of ADMA (Fig. 6).
Discussion
NO, an important local regulator factor in cardiovas-
cular tissues, is synthesized from l-arginine by NOS in
endothelial cells. It possesses complex cardiovascular
actions such as protecting endothelial cells, decreasing
the endothelial adhesiveness and inhibiting the adhesion
Figure 4. Effect of xanthone on levels of MCP-1 in the conditioned
medium. Xan: 1,3,5,6-tetrahydroxyxanthone. MeansꢁSEM, n=6.
Figure 6. Effect of xanthone on levels of ADMA in the conditioned
medium. Xan: 1,3,5,6-tetrahydroxyxanthone. MeansꢁSEM, n=6.
++
p<0.01 versus control, *p<0.05, **p<0.01 vs ox-LDL.
++
p<0.01 versus control, *p<0.05, **p<0.01 versus ox-LDL.
Figure 5. Effect of xanthone on concentrations of nitrite/nitrate in the
conditioned medium. Xan: 1,3,5,6-tetrahydroxyxanthone. Mean-
Figure 7. Effect of xanthone on activity of endothelial DDAH. Xan:
++
++
sꢁSEM, n=6.
p<0.01 vs control, *p<0.05, **p<0.01 vs ox-
1,3,5,6-tetrahydroxyxanthone. MeansꢁSEM, n=6.
p<0.01 versus
LDL.
control, *p<0.05, **p<0.01 versus ox-LDL.

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D.-J. Jiang et al. / Bioorg. Med. Chem. 11 (2003) 5171–5177
of monocytes to endothelial cells.1
4ꢀ16
Recently, it has
DDAH in cultured endothelial cells.13,31 These results
suggest the increased level of ADMA induced by ox-
LDL may be related to decrease of DDAH activity by
elevation of TNF-a production.
been found that l-arginine analogues such as ADMA,
which is present in blood of both humans and animals,
can inhibit NOS in vivo and in vitro.
1
7,18
There is
growing evidence that endothelial dysfunction in some
cardiovascular diseases such as hypercholesterolemia,
heart failure and hypertension is associated with eleva-
tion of ADMA, and endogenous inhibitors of NOS
have been thought as a novel predictor of endothelial
Xanthones, a kind of ployphenolic compound, have
extensive pharmacological actions including antioxida-
tion, anti-inflammation and cardioprotection.
1
,5,6
Recently, it was reported that some synthetic xanthones
significantly inhibited the increased expression of
ICAM-1 induced by TNF-a in cultured human endo-
1
9
dysfunction. ADMA is synthesized by protein arginine
methyltransferases (PRMTs), which utilizes S-adeno-
sylmethionine methyl groupdonor, and degraded by
dimethylarginine dimethylaminohydrolase (DDAH),
which hydrolyzes ADMA to l-citrulline and dimethy-
4
thelial cells. Our recent work has also shown that
daviditin A, a xanthone compound extracted from
S. davidi Franch (Gentianaceae), protects the endothe-
lium against damage induced by LPC concomitantly
1
3,20
lamine asymmetric dimethylarginine.
ADMA and
7
DDAH are widely distributed in tissues including endo-
with a decrease of ADMA level. The results of the
21
thelial cells. There is evidence that lipid-induced dysre-
gulation of DDAH may be an important factor
contributing the elevation of ADMA in hypercholestero-
laemia and hyperhomocyst(e)inemia.9,22 Others have
been reported that the elevated content of ADMA is
also related the decreased activity of DDAH in cultured
present study revealed that xanthone significantly
inhibited the release of LDH, decreased the level of
MCP-1 and attenuated the increased adhesion of
monocytes to endothelial cells in cultured endothelial
cells induced by ox-LDL. As mentioned above, there
are interactions of ADMA with cytokines in the devel-
opment of atherosclerosis. In the present study xan-
thone also significantly attenuated the elevations of
TNF-a and ADMA level and decrease of DDAH
activity in cultured endothelial cells treated with ox-
LDL, suggesting that the protective effect of xanthone
on endothelial cells may be related to reduction of
ADMA level.
1
3
endothelial cells treated with LDL or ox-LDL.
There is growing evidence that ox-LDL plays a key role
2
3
in the development of atherosclerotic lesions. Ox-LDL
exhibits numerous biological effects, including endothe-
lial dysfunction, activation of endothelial adhesiveness,
2
4,25
monocyte differentiation and adhesion.
As has been
1
3
reported previously, in the present study ox-LDL sig-
nificantly increased the release of LDH which is positive
related to the extent of cell injury, and decreased the
activity of DDAH and increases the level of ADMA in
the cultured endothelial cells. It has been shown that the
level of ADMA is associated with an increase in adhe-
siveness of monocyte in patients with hypercholester-
Conclusion
The present results suggest that xanthone preserves the
damage of endothelial cells and inhibits the addition of
monocytes to endothelial cells induced by ox-LDL, and
that the protective effect of xanthone on the endothelial
cells is related to decrease of ADMA concentration via
increasing DDAH activity.
2
6
olemia. In the present study, treatment with ox-LDL
increased the expression of MCP-1 and the adhesion of
monocytes to endothelial cells concomitantly with an
increase level of ADMA. Others have found that
ADMA enhances adhesion of monocytes in cultured
2
7
endothelial cells, in further support of the hypothesis
that the increased adhesion of monocytes to endothelial
cells induced by ox-LDL is related to elevation of
ADMA level.
Experimental
Materials
Probucol, asymmetric dimethylarginine (ADMA) was
obtained from Sigma. DMEM was obtained from
Gibco. Lactate dehydrogenase (LDH) and nitric oxide
(NO) assay kits were obtained from Ju-Li Biological
Medical Engineering Institute (Nanjing, P. R. China).
TNF-a and monocyte chemotactic protein-1 (MCP-1)
assay kits were obtained from Sen-xiong science and
technology industrial Co. Ltd (Shanghai, China).
It has been suggested that inflammatory responses are
8
involved in the development of atherosclerosis. It was
reported that inflammatory cytokines such as TNF-a
were significantly increased in animals and patients with
2
8,29
hypercholesterolaemia,
lated the expression of adhesion molecules and
and that TNF-a upregu-
3
0
increased the adhesion of monocyte to endothelium,
suggesting to play an initial factor in a series of inflam-
matory response in the atherosclerotic lesions. TNF-a is
present in various cells including macrophages and
endothelial cells. Recently, it has been shown that
treatment with TNF-a significantly elevated the level of
ADMA concomitantly with a decrease of DDAH
activity in cultured human endothelial cells. The pres-
ent results confirmed previous observations that treat-
ment with ox-LDL significantly increased the levels of
TNF-a and ADMA and decreased the activity of
Synthesis of xanthone
1,3,5,6-Tetrahydroxyxanthone was synthesized by the
Friedel-Crafts acylation of 1,3,5-trimethoxybenzene
with 2,3,4-trimethoxybenzoyl chloride to yield a mixture
1
3
0 0 0
of 2-hydroxy- 2 ,3,4,4 ,6 -pentamethoxybenzophenone
0
0
0
and 6 -hydroxy-2,2 ,3,4,4 -pentamethoxybenzophenone
which cyclised to give 1,3,5,6-tetra-hydroxyxanthone,
3
2 1
followed by demethlation (Fig. 8).
NMR: d 6.20 (d,

D.-J. Jiang et al. / Bioorg. Med. Chem. 11 (2003) 5171–5177
5175
J=2.5 Hz, 1H, H-2), 6.46 (d, J=2.5 Hz, 1H, H-4), 6.91
d, J=8.5 Hz, 1H, H-7), 7.57 (d, J=8.5 Hz, 1H, H-8);
literature data: NMR: d 6.28 (d, J=2.5 Hz, 1H, H-2),
Monocyte-endothelial cell adhesion assay
(
1
Monocytes were obtained from fresh peripheral blood
of healthy volunteers. Monocytes were isolated by the
Ficoll–Paque density centrifugation method as pre-
6
7
.54 (d, J=2.5 Hz, 1H, H-4), 6.99 (d, J=8.5 Hz, 1H, H-
), 7.96 (d, J=8.5 Hz, 1H, H-8) Xanthone was initially
3
6
dissolved in ethanol and further diluted in conditioned
medium to proper final concentration.
viously described.
The collected monocytes were
washed twice in Hanks’ balanced salt solution (HBSS)
and resuspended in DMEM containing 10% (v/v) FBS,
1
00 U/mL penicillin and 100 mg/mL streptomycin. This
Preparation and oxidation of LDL
preparation method routinely harvested >95% of
monocytes and maintained >95% viability as assessed
by trypan blue exclusion.
Native LDL (nLDL) was isolated from freshly prepared
normal human plasma though sequential density gra-
dient ultracentrifugation in sodium bromide density
solutions in the density range 1.019–1.063 g/mL as pre-
viously described.33 Then LDL was dialyzed against
The adhesion of the monocytes to endothelial cells was
evaluated by protein content as previously described
methods. The monolayers of endothelial cells treated
were washed three times with HBSS before addition of
monocytes. Monocytes cells were diluted to a final con-
3
7
0
.01 M PBS (0.01 M phosphate buffer, 2.7 mM KCl, 137
mM NaCl, pH 7.4) containing 0.01% EDTA. Protein
concentration was measured by previously described
3
4
6
methods.
centration of 10 cells/mL and were added to each well
(1 mL) of endothelial cells. An additional 1 mL of the
Oxidation of nLDL was induced by adding 10 mM
monocyte suspension was also obtained for protein
determination. The monocytes were allowed to incubate
with the endothelial cells monolayer at room tempera-
ture for 30 min on a rocking platform and nonadherent
monocytes were carefully removed by two washes with
HBSS. ECV304 cells and adherent monocytes were then
lysed with 0.5 M NaOH for protein analysis as pre-
ꢂ
CuSO for 24 h at 37 C. The amount of thiobarbituric
4
acid reactive substance (TBARS), reflecting the extent
of LDL oxidation, was measured by previously descri-
3
5
bed methods. The amount of TBARS was 6.80ꢁ0.92
and 24.19ꢁ4.79 nmol/mg protein for nLDL and
oxLDL, respectively (n=3, p<0.01).
3
4
viously described. The adhesion of monocytes was
estimated by comparing the amount of protein in wells
containing endothelial cells and monocytes minus the
the amount of protein in wells containing ECV304 cells
alone, divided by the amount of protein in 1 mL of
monocytes suspension.
Cell culture and treatment
ECV304 cells, a human umbilical vein endothelial cell
line, were cultured in Dulbecco’s modified Eagle’s med-
ium (DMEM) containing 10% (v/v) fetal bovine serum
(
FBS), 100 U/mL penicillin and 100 mg/mL streptomy-
cin. When the cells had reached subconfluence, cells
were passaged into 24-well culture dishes and the cul-
ture medium was replaced the serum-free medium. Cell
Determination of TNF-ꢀ concentration
TNF-a level in culture medium was assayed by enzyme-
linked immunosorbent assay (ELISA). The values were
measured at 405 nm by a microplate reader (Biotek).
The standard curve for TNF-a measured by this ELISA
was linear from 78 pg/mL to 10 ng/mL; the detection
limit was 100 pg/mL.
6
numbers were 6.1ꢁ0.3ꢃ10 cells/well (n=6). Cells were
counted by trypan blue exclusion and showed >95%
viability. Cells were treated with ox-LDL. For xanthone
and probucol which was as a positive control, endothe-
lial cells were exposed to xanthone (1, 3 or 10 mmol/L)
or probucol (5 mmol/L) for 1 h, and then exposed to ox-
LDL for 48 h in the presence of xanthone or probucol.
Determination of MCP-1 concentration
MCP-1 in culture medium was measured by ELISA
using with recombinant human MCP-1 as a standard.
The values were measured at 492 nm by a microplate
reader (Biotek). The standard curve for MCP-1 mea-
sured by this ELISA was linear from 8 to 500 pg/mL;
the detection limit was 10 pg/mL.
Determination of LDH activity
The activity of LDH in the conditioned medium, as an
indicator of cell cytotoxicity, was measured spectro-
photometrically using a commercially available assay
kit.
Determination of nitrite/nitrate concentration
The level of nitric oxide in the conditioned medium was
determined indirectly as the content of nitrite and
nitrate. Level of nitrite/nitrate in the cells conditioned
3
8
medium was measured as previously described.
Determination of ADMA concentration
The protein in the conditioned medium was removed
using 5-sulfosalicylic acid (5-SSA). The content of
Figure 8. The synthesis of 1,3,5,6-tetrahydroxyxanthone.

5176
D.-J. Jiang et al. / Bioorg. Med. Chem. 11 (2003) 5171–5177
ADMA was measured high-performance liquid chro-
matography (HPLC) as described previously with some
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3
9
modification. HPLC was carried out using a Shi-
madzu LC-6A liquid chromatograph with Shimadzu
SCL-6A system controller and Shimadzu SIC-6A auto-
sampler. O-Phthaldiadehyde adducts of methylated
amino acids and internal standard ADMA produced by
precolumn mixing were monitored using a model RF
6
. Jiang, D. J.; Tan, G. S.; Ye, F.; Du, Y. H.; Xu, K. P.; Li,
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30 fluorescence detector set at l =338 and l =425
¨
¨
nm on a Resolve C18 column. Samples were eluted from
the column using a linear gradient containing mobile
phase A composed of 0.05 mol/l (pH 6.8) sodium ace-
tate–methanol–tetrahydrofuran (81:18:1 v:v:v) and
mobile phase B composed of 0.05 mmol sodium acetate–
methanol–tetrahydrofuran (22:77:1 v:v:v) at a flow-rate
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DDAH activity assay
1
The activity of DDAH in endothelial cells was esti-
mated by directly measuring the amount of ADMA
metabolized by the enzyme. In an ice bath, cell lysates
were divided into two groups, and ADMA was added
4
0
4
1
202.
6. Hegarty, N. J.; Young, L. S.; Kirwan, C. N.; O’Neill, A. J.;
(
3
final concentration 500 mmol/L). To inactivate DDAH,
0% sulfurosalicylic acid was immediately added to 1
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0
3
% DDAH activity. The other lysate was incubated at
ꢂ
7 C for 2 h before the addition of 30% sulfurosalicylic
1
acid. The ADMA level in each groupwas measured by
HPLC as described above. The difference in ADMA
concentration between two groups reflected the DDAH
activity. For every experiment, DDAH activity of
ECV304 cells exposed to normal conditioned medium is
defined as 100%, and DDAH activity in other condi-
tions were expressed as percentages of the ADMA
metabolized compared with the control.
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2
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