-induced endothelial
cells in vitro1 Ma Y et al / Acta Pharmacol Sin 2004 Feb; 25 (2): 246-250
-induced endothelial
cells in vitro1
Yi MA, Jian-ning SUN2, Qiu-ping XU, Zi-li YOU3, Ya-jian GUO
Department of Pharmacology, Beijing University of Chinese Traditional Medicine, Beijing 100102; 3School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China
1 Project supported by the State Science and Technology Commission grant, No 2001BA701A07-14.
2 Correspondence to Prof Jian-ning SUN. Phn 86-10-6471-1199 ext 6084. Fax 86-10-6472-1242. E-mail JN_SUN @sina.com
Received 2002-11-22 Accepted 2003-06-20
KEY WORDS 3,4-oxo-isopropylidene-shikimic acid; adhesions; vascular endothelium; neutrophils; intercellular adhesion molecule-1
ABSTRACT
AIM: To examine the effect of 3,4-oxo-isopropylidene-shikimic acid (ISA)
on human polymorphonuclear leukocyte (PMN) adhesion to human umbilical vein
endothelial cells (HUVEC) and explore its mechanism. METHODS: Adhesion
of PMN to HUVEC was measured by rose bengal staining assay. Cell-ELISA and RT-PCR
methods were used to examine the expression of adhesion molecules ICAM-1. Cell
viability was detected with MTT assay. RESULTS: ISA (1-100 µmol/L)
effectively reduced PMN adhesion to TNF--induced
HUVEC with the inhibitory rate from 17.2 % to 53.5 %, and exerted no effect
on PMN adhesion to normal HUVEC. Adhesion molecule ICAM-1 surface protein and
mRNA expression induced by TNF-
(400 kU/L) were significantly inhibited by ISA. In addition, the cell viability
of HUVEC was unchanged 48 h after treatment with ISA. CONCLUSION: ISA
inhibited TNF--stimulated PMN-HUVEC
adhesion and expression of ICAM-1.
INTRODUCTION
Leukocyte infiltration to endothelium plays a major role in the inflammatory
response related to thrombosis, arteriosclerosis, and reperfusion injury, etc.
Leukocyte infiltration requires a chain of reactions between endothelial cells
(EC) and leukocytes that initially retards intravascular leukocyte flow and
finally leads to leukocyte transmigration through the endothelial monolayer.
These events are mediated by sequential interaction of different endothelial
adhesion molecules with their receptors on leukocyte surface. A crucial step
between the initial contact and final transmigration of leukocytes is their
tight adhesion to EC, which is mainly mediated by the endothelial transmembrane
receptors intercellular adhesion molecule-1 (ICAM-1, CD54) and vascular adhesion
molecule-1 (VCAM-1, CD106). The up-regulation of ICAM-1 on the surface of the
endothelium is required for the firm adhesion of rolling polymorphonuclear leukocyte
(PMN). Lipopolysaccharide (LPS) and inflammatory cytokines, including tumor
necrosis factor- (TNF-),
interleukin-1(IL-1), and interferon-
can stimulate ICAM-1 expression[1,2]. In this study, TNF-
was used to stimulate human umbilical vein endothelial cells (HUVEC) expression
adhesion molecule and induced PMN-endothelial cell adhesion. Based on this model,
the effect of 3,4-oxo-isopropylidene-shikimic acid (ISA) on adhesion was observed.
ISA was one of the derivatives of shikimic acid (SA), which was extracted from Illicium verum Hookfil. Our previous study suggested that ISA could suppress various experimental thrombosis induced by injuring vascular endothelium[3] and attenuate cerebral ischemic damage[4,5]. To further clarify the above action of ISA, the effect of ISA on PMN-endothelial cell adhesion was observed and its mechanism was explored.
Structural formula of 3,4-oxo-isopropylidene-shikimic acid (ISA)
MATERIALS AND METHODS
Chemicals ISA (purity >98 %), provided by Department of Phytochemistry, Beijing University of Traditional Chinese Medicine, was dissolved in pure water and kept as a stock solution. RT-PCR primer was synthesized by Shanghai Sangon Co. 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide was purchased from Sigma. Freshly discarded human umbilical cords were obtained from China-Japan Friendship Hospital.
Cell culture Human umbilical vein endothelial cells (HUVEC) were harvested from umbilical cords by collagenase treatment as described by Jaffe et al[6]. The cells were grown to confluence in medium 199 (Gibco) supplemented with 20 % fetal bovine serum (Hyclone), benzyl penicillin (100 kU/L), streptomycin (100 mg/L), glutamine (2 mmol/L) and endothelial cell growth factor (20 mg/L, Roche ) in culture flasks and dishes coated with 1 % gelatin. To confirm the endothelial origin of the harvested cells, we analyzed them under microscope for the typical cobblestone appearance and tested immunohistochemically for the occurrence of von Willebrand factor. For experiments, the second or third passage of cells was used.
PMN isolation Human peripheral blood PMN were obtained from healthy adult volunteers and separated on a discontinuous gradient consisting of Ficoll-Hypaque solution with the density of 1.077 kg/L and 1.119 kg/L[7,8]. Isolated PMN were resuspended in M199 at a final concentration of 3×109 cells/L. PMN purity exceeded 98 % as confirmed by Wright's stain. Cell viability exceeded 95 % by trypan blue exclusion.
Adhesion assays PMN were added to HUVEC
monolayers at a PMN-to-HUVEC ratio of 10:1. After
coincubation for 40 min, the wells were gently rinsed
twice with PBS to remove nonadherent cells. PMN adhesion to endothelial cells was evaluated by rose bengel
staining[9]. Color development was measured with a
microtiter plate spectrophotometer (Multiskan MK3,
Thermo Co) at 570 nm and subtracted
A570nm of wells containing EC alone to indicate the adhesion of PMN.
Each adhesion assay was performed 12 h after treating
monolayers with TNF-. The effect of ISA on
PMN adhesion was assessed by pre-incubation of ISA
with monolayers for 6 h before adding
TNF-.
Cell ELISA for ICAM-1[10]
HUVEC were seeded in 96-well tissue culture dishes with
3×104cells/well. Treatment of HUVEC monolayers with ISA or
TNF- was the same as described in adhesion assays.
After stimulation with TNF- for 12 h, the cell
culture medium was removed, and the cells were immediately fixed with methanol. Nonspecific protein
binding was blocked subsequently by adding 1 % BSA in
PBS for 40 min at 37 ºC. Primary mouse monoclonal
antibodies for ICAM-1 (NeoMarkers) were added to each well (1:1000) and incubated at 37 ºC for 1 h. The
cells were washed in PBS-0.05 % Tween, and incubated at 37 ºC for 1 h with horseradish
peroxidase-conjugated goat anti-mouse IgG (Zhongshan Co, 1:10
000). The cells were washed again with PBS-0.05 %
Tween three times, and the solution of substrate
(citrate-phosphate buffer containing 0.04 % OPD, Sigma) was
added for 15 min. The reaction was stopped with
sulfuric acid 2 mol/L (50 µL), and the color development
was read on a microtiter plate spectrophotometer (Multiskan MK3, Thermo Co) at 492 nm after
subtracting the background values in cells stained only with the
second-step antibody.
ICAM-1 mRNA expression with RT-PCR Semiquantitative PCR was used to assess ICAM-1
expression. After incubated with ISA, the cells were
treated for 6 h with TNF- (400 kU/L). Total
RNA of HUVEC was extracted with Trizol reagent
(Invitrogen) according to the manufacturer's instruction. Quantification and purity of RNA were
assessed by
A260/A280 absorption. The RNA samples
with A260/A280 ratios (above 1.9) were used for further
analysis. First-strand cDNA was synthesized from the
total RNA by Omniscript RT (Qiagen) following the manufacturer's instructions. cDNA was amplified by
PCR in a total volume of 50 µL using 2.5 U
Taq DNA polymerase (Promega) and 10 pmol each of
upstream and downstream primers. After predenaturation
at 94 ºC for 3 min, 35 cycles were allowed to run for
30 s at 94 ºC, followed by 30 s at 55 ºC and 30 s at 72
ºC, and a final extension at 72 ºC for 10
min. Primers for ICAM-1 were sense 5'-AAT GCC CAG ACA TCT
GTG TCC C-3', antisense 5'-GGC AGC GTA GGG TAA GGT TCT T-3', and for GAPDH, sense 5'-TGG TAT
CGT GGA AGG ACT CAT G-3, antisense 5'-TCC TTG GAG GCC ATG TGG GCC
AT-3'. The predicted amplification products were 330 bp and 501 bp,
respectively. A 15-µL aliquot of the amplified
DNA reaction mixture was fractionated by 1.5 %
agarose gel electrophoresis, and the amplified product was
then visualized by ultraviolet fluorescence after being
stained with ethidium bromide. ICAM-1 mRNA expression was normalized to the housekeeping gene GAPDH
mRNA expression via densitometric analysis by autogel
analysis system. The results were expressed as the
relative level of mRNA expression (ratio of
ICAM-1/GAPDH).
Cell viability assays Colorimetric 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to determine cell viability[12] 48 h after ISA treatment .
Statistical analysis Each experiment was performed in triplicate and repeated at least three times. Data were presented as mean±SD. Differences were analyzed by ANOVA .
RESULTS
Effect of TNF- on adhesion
of PMN to HUVEC PMN adhesion to HUVEC was increased by preincubation of
HUVEC with TNF- (100-800 kU/L)
for 12 h, in a concentration-dependent manner (Fig 1).
Fig 1. Effect of TNF- on adhesion of PMN to HUVEC. n=3. Mean±SD.
bP<0.05, cP<0.01
vs control.
Effects of ISA on the adhesion of PMN to TNF--stimulated
and normal HUVEC PMN adhesion to HUVEC was increased by incubation of HUVEC
with TNF- (400 kU/L) for 12 h
(P<0.01 vs control) . Pretreatment with ISA 1-100 µmol/L
inhibited PMN adhension to TNF--induced
HUVEC in a concentration-dependent manner, with the inhibitory rate from
17.2 % to 53.5 %. Treatment of EC only with ISA for 18 h exerted no effect on
PMN adhension to HUVEC compared with control (Tab 1).
Tab 1. Effect of ISA on PMN adhesion to TNF--induced HUVEC and normal HUVEC.
n=3. Mean±SD. aP>0.05, bP<0.05,
cP<0.01 vs control. eP< 0.05,
fP< 0.01 vs TNF- group.
ISA: 3,4-oxo-isopropylidene-shikimic acid; HUVEC: human umbilical vein endothelial cells.
Effect of ISA on ICAM-1 surface protein expression in HUVEC ICAM-1 was
expressed at low levels on untreated HUVEC and up-regulated by stimulation with
TNF- (400 kU/L) for 12 h. Pretreatment
with ISA (10 and 100 µmol/L) attenuated TNF--induced
ICAM-1 expression in HUVEC, the inhibitory rate was 19.7 % and 40.4 %, respectively
(Tab 2).
Tab 2. Inhibitory effects of ISA on expression of ICAM-1 surface protein
of HUVEC induced by TNF- (400 kU/L ) by cell-ELISA method. n=3.
Mean±SD. bP<0.05, cP<0.01 vs
control. eP<0.05, fP<0.01 vs
TNF- group.
ISA: 3,4-oxo-isopropylidene-shikimic acid; HUVEC: human umbilical vein endothelial cells.
Effect of ISA on ICAM-1 mRNA expression in HUVEC The products of RT-PCR
for ICAM-1 mRNA and GAPDH mRNA were 330 bp and 501 bp respectively, corresponding
to the predicted length (Fig 2). In control group, a basic expression of ICAM-1
in HUVEC was found. TNF- (400
kU/L) enhanced expression of ICAM-1 mRNA in HUVEC significantly. Pre-treatment
with ISA (10 µmol/L) obviously decreased the expression level of ICAM-1
mRNA induced by TNF- (P<0.05).
Fig 2. The inhibitory effects of ISA on ICAM-1 mRNA expression in cultured
HUVEC stimulated with TNF- (400 kU/L) for 6 h. A) ICAM-1 (330 bp) and GAPDH
( 501 bp ) mRNA levels in cultured HUVEC were detected by RT-PCR. The samples
were loaded on 1.5 % agarose gel. Lane 1: TNF-+ISA 10 µmol/L; Lane 2:
TNF-; Lane 3: control; Lane 4: 100-bp DNA ladder. B) ICAM-1 mRNA level was
expressed as the ratio of ICAM-1 mRNA relative to the GAPDH mRNA levels.
n=4. Mean±SD. cP<0.01 vs
control group. eP< 0.05 vs TNF-
group.
Effect of ISA on cell viability of HUVEC There was no difference in cell viability between HUVEC incu-bated with ISA for 48 h and untreated HUVEC (Tab 3).
Tab 3. Effect of ISA on cell viability of HUVEC. n=3. Mean±SD. aP>0.05 vs control.
ISA: 3,4-oxo-isopropylidene-shikimic acid; HUVEC: human umbilical vein endothelial cells.
DISCUSSION
Adhesion of circulating PMN to the vascular endothelium is a critical step
in the inflammatory response related to thrombosis, arteriosclerosis, and reperfusion
injury, etc. Our previous study showed that ISA could suppress various
experimental thrombosis and attenuate ischemic injury. Whether this effect is
mediated by inhibition of adhesion of PMN to vascular endothelium is not clear.
The present results showed that ISA effectively reduced PMN adhesion to TNF--induced
HUVEC in a concentration-dependent manner and exerted no effect on PMN adhesion
to normal HUVEC. To exclude the decrease of PMN adhesion to HUVEC for the cytotoxicity
induced by ISA, cell viability of HUVEC after treatment with ISA for 48 h was
observed. The result showed the cell viability unchanged. So we concluded that
ISA inhibited PMN adhesion to TNF--induced
HUVEC not for its cytotoxicity to HUVEC.
To explore the inhibitory mechanism of ISA on PMN adhesion to HUVEC, the surface protein and mes
senger RNA expression of ICAM-1 were examined. The
up-regulation of ICAM-1 on the surface of the
endothelium is required for the firm adhesion of rolling
PMN[12]. ICAM-1, a member of the immunoglobulin supergene
family, serves as the receptor for leukocyte
function-associated antigen-1 (LFA-1; CD11a/CD18),
2-integrin, which is expressed on PMN, monocytes,
lymphocytes, and natural killer
cells[1]. ICAM-1 is constitutively expressed at low levels on the cell surface of
endothelial and epithelial cells, and up-regulated fast
after stimulation with proinflammayory
cytokine[13], which is accordance with our study. In our study,
TNF- increased PMN-endothelial cell adhesion in a
concentration-dependent manner and upregulated ICAM-1
expression and ICAM-1 mRNA levels in HUVEC. ISA was found to attenuate
TNF--induced ICAM-1 expression at the dose of 10
µmol/L, but ISA 1 µmol/L could inhibit PMN adhesion to
TNF- -induced HUVEC. These all suggested that the inhibitory effect
of ISA on PMN-EC adhesion was not only associated with inhibitory expression of ICAM-1, but also with
other effects, which needs further study. ISA also
inhibited TNF--induced ICAM-1 mRNA
expression in HUVEC, which indicated the mechanism of ISA
anti-adhesion at least in part related to down-regulation
of the gene transcription of ICAM-1.
Taken together, the results suggested that suppressing the surface protein and mRNA expression of
ICAM-1 was one of the pathways of ISA inhibiting adhesion of PMN to
TNF--stimulated HUVEC.
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