Intern
ati
o
n
a
l
Jo
urn
a
l
o
f
P
u
b
lic Hea
l
th Science (IJ
P
HS)
V
o
l.4
,
No
.3
, Sep
t
em
b
e
r
20
15, pp
. 205
~210
I
S
SN
: 225
2-8
8
0
6
2
05
Jo
urn
a
l
h
o
me
pa
ge
: h
ttp
://iaesjo
u
r
na
l.com/
o
n
lin
e/ind
e
x.ph
p
/
IJPHS
High Gl
ucos
e, but Not T
e
st
ost
e
rone, In
creas
e
s Pl
at
elet
Aggregation Mediated
by Endothelial Cells
Ikhlas
Muh
a
mmad
Je
nie
1
, Budi
Mulyon
o
2
, Soedj
o
n
o
As
w
i
n
3
, Sri Ka
da
rsih So
ejono
4
1
Department of Ph
y
s
iolog
y
,
Faculty
of
Medicine and H
ealth Scie
nces,University
Muhammadiy
a
h Yog
y
ak
arta,
Ind
onesia
2
Department of Clinical
Patholo
g
y
,
Facu
lty
of
M
e
dicine, Gadjah
Mada
University, Indonesia
3
Department of Anatom
y
and Embriolo
g
y
Department, Faculty
of Medicine
, Gadjah Mad
a
Univ
ersity
, Indonesia
4
Department of Ph
y
s
iolog
y
, Faculty
of
Medicine, Gadjah Mad
a
University
,
Indo
nesia
Article Info
A
B
STRAC
T
Article histo
r
y:
Received Aug 1, 2015
Rev
i
sed
Au
g
20
, 20
15
Accepted Aug 26, 2015
Endothelial cells inhi
bit p
l
atelet
aggregation b
y
r
e
leasing thromb
oregulators,
such as prosta
c
y
clin
and n
itri
c
o
x
ide.
Ma
le
subje
c
t is
a
tradi
tiona
l risk f
acto
r
for card
i
ovas
c
ul
ar dis
e
as
es
. P
l
a
t
el
et h
y
p
e
rre
act
i
v
it
y has
b
een
frequen
t
l
y
found in patient
with diab
etes mellitus.
To
examine whether
testo
s
terone an
d
high glucose modif
y
p
l
atelet agg
r
egati
on through
endothelial
cells, we did an
in vitro stud
y
u
s
ing endothel
i
al
cells cu
ltur
e
fr
om
hum
an u
m
b
ilical v
e
in
(HUVEC).
Treatments were performed
in HU
VEC sub culture as either
normoglucose (5
.6 mM) or high
glucos
e (22
.
4 m
M
) medium, with or withou
t
testosteron
e
(0,
1, 10, 100 nM)
,
fo
r 24 ho
urs.
HUVEC were tr
y
p
sinized
,
res
u
s
p
ended,
an
d then
incub
a
t
e
d
with p
l
at
ele
t
r
i
c
h
plas
m
a
from
h
eal
th
y m
a
le
donors with ratio 1:10
4
for 3 minutes. Platelet aggregation measured
b
y
turbidimetr
y
methode. This stud
y
s
howed that testosterone did no
t
significantly
inf
l
uence platelet a
ggregation th
rough endothelial cells in
normoglucose (
p
= 0.144) or high
glucose (
p
= 0
.
9
16) medium. There was no
main effect of testosterone (
p
= 0.73)
as well as
no inter
a
ction
between
testosteron
e
and
glucose (
p
= 0.6
9
), but
there was a main eff
e
ct of
glucose (
p
= 0.004), to platelet aggr
egatio
n thr
ough endothelial cells. In
conclusion
,
high glucos
e, bu
t not
testosteron
e
,
inhibi
ts
pla
t
e
l
et aggrega
tion
m
e
diated
b
y
endothelial cells.
Keyword:
End
o
t
h
e
lial cells
Hype
rglycem
ia
In v
itro
Platelet aggre
g
ation
Test
ost
e
r
one
Copyright ©
201
5 Institut
e
o
f
Ad
vanced
Engin
eer
ing and S
c
i
e
nce.
All rights re
se
rve
d
.
Co
rresp
ond
i
ng
Autho
r
:
Sri Kad
a
rsih So
ejon
o,
Depa
rt
m
e
nt
of
Phy
s
i
o
l
ogy
,
Gad
j
a
h
M
a
da
Uni
v
ersity
,
Jl
. Farm
ako,
S
e
ki
p
Ut
ara
,
Yo
gy
aka
r
t
a
5
5
2
8
1
,
I
n
d
o
n
esi
a
.
Em
a
il: ik
h
l
asj
e
n
i
e@yahoo
.co
.
u
k
1.
INTRODUCTION
End
o
t
h
e
lial cells (EC) form
co
n
tinu
e
s m
o
no
layer ep
ith
eli
a
l cells l
i
n
i
n
g
th
e in
ternal lu
men
o
f
th
e
bl
o
o
d
ve
ssel
s
.
In
n
o
rm
al
phy
s
i
ol
ogi
cal
c
o
ndi
t
i
on,
EC
,
bei
n
g
ex
po
sed
t
o
bl
oo
d
gl
uc
ose
l
e
vel
s
i
n
ra
n
g
e
3
.
6
-
5
.
8
mmo
l/L, is characterized as
an
tith
ro
m
b
o
tic and
an
tico
a
gu
lan
t
. Howev
e
r,
wh
en EC are
b
e
ing
exp
o
sed
t
o
hy
pe
rgl
y
cem
i
a
, as t
h
e hal
l
m
a
rk o
f
di
a
b
et
es m
e
l
l
i
t
u
s (DM
)
,
cel
l
ho
m
e
ost
a
si
s i
s
pert
ur
bed
and EC
m
a
y
chan
g
e
t
o
be
pr
ot
h
r
om
bot
i
c
a
n
d
pr
oc
oag
u
l
a
nt
.
E
n
d
o
t
hel
i
a
l
dy
sf
unc
t
i
on i
s
bel
i
e
ve
d t
o
occu
r
bef
o
re
t
h
e
de
vel
o
pm
ent
of m
i
crovasc
u
l
ar an
d m
acrovasc
ul
ar c
o
m
p
l
i
cat
i
ons i
n
t
y
pe
2 di
a
b
et
es
m
e
l
l
i
t
u
s (T
2
D
M
)
pat
i
e
nt
s.
The
unde
rlying m
e
chanism
includes the accel
erated form
ation of advance
d
gl
ycation end product and activation
of
p
r
ot
ei
n
ki
na
se C
[
1
]
-
[
2
]
.
In recent years
,
testosterone (T) gets m
o
re attenti
on as a potential player in cardi
ova
scular diseas
e
s
(CVD), alt
h
oug
h
m
a
le sex
has b
e
en
reco
gn
ized
as a trad
itio
n
a
l risk
facto
r
in
C
V
D for a lon
g
ti
me ag
o.
Androge
n
rec
e
p
tor (AR) a
n
d
enzym
e
s that metabolizes T
are found in
E
C
and the
rest
of cardiovasc
u
lar (C
V)
sy
st
em
[3]
-
[
4
]
.
T m
a
y
affect
EC
by
ge
n
o
m
i
c an
d n
o
n
-
g
e
n
om
i
c
pat
h
way
.
Ho
we
ver
,
t
h
e
resul
t
s
of t
h
e s
t
udi
es
Evaluation Warning : The document was created with Spire.PDF for Python.
I
S
SN
:
2
252
-88
06
I
J
PH
S Vo
l. 4
,
N
o
. 3
,
Sep
t
emb
e
r
201
5
:
2
05
–
21
0
20
6
in
th
is area are still
co
n
f
lictin
g
.
Fo
r ex
am
p
l
e, as th
e le
v
e
l
o
f
seru
m
T is
rath
er low in
patien
t
s with
CVD, as
well as in aging m
e
n, T re
placem
ent offers a potentia
l bene
fit but a
b
rupt uprising of T level, s
u
ch as
expe
ri
ence
d
by
at
hl
et
es w
h
o t
a
ke a
n
d
r
oge
n,
i
s
associ
at
e
d
wi
t
h
C
V
eve
n
t
s
[
5
]
-
[
7
]
.
In CV ev
en
ts,
p
l
atelet ag
gregatio
n
can
b
e
reg
a
rd
ed
as th
e early ev
en
t t
h
at will lead
t
o
th
ro
m
b
o
s
is
form
at
io
n
[8
].
Fo
rt
un
ately, EC will resp
on
se to
p
l
atel
et activ
atio
n
b
y
releasin
g
t
h
ro
m
b
o
r
egu
l
ato
r
s, such
as
p
r
o
s
tacyclin
an
d n
itric ox
id
e [9
]. Th
us, it i
s
v
e
ry in
terestin
g to
ex
am
in
e wh
eth
e
r t
h
e
cap
acity o
f
EC was
chan
ge
d a
f
t
e
r e
x
p
o
s
u
re
o
f
T
i
n
n
o
rm
ogl
uc
ose
(N
G
)
or
hi
gh
gl
uc
ose
(H
G
)
e
nvi
ro
nm
ent
.
2.
R
E
SEARC
H M
ETHOD
Th
e stud
y was
ap
pro
v
e
d
b
y
the In
stitu
tion
a
l
Rev
i
ew Bo
ard
o
f
t
h
e Facu
lty o
f
Med
i
cin
e
Gad
j
ah
Mad
a
Un
i
v
ersity (FM GMU), Yog
y
ak
arta. Dono
rs
for
u
m
b
ili
cal co
rd
s (n
= 2
)
were m
o
thers who
d
e
liv
ered
h
e
r
b
a
b
y
in
a m
i
d
w
ifery clin
ic or Mu
h
a
mm
ad
i
y
ah
’s ho
sp
i
t
a
l
s
i
n
Yo
gy
aka
r
t
a
. The m
o
t
h
ers had
no
hy
pe
rt
ensi
o
n
,
diabetes m
e
llitus, and (pre
) eccla
m
psia, the delivery wa
s a
t
erm
,
and the
baby wa
s havi
ng Apgar
sc
ore
>10.
Do
n
o
rs f
o
r pl
a
t
el
et
s (n = 2) were
heal
t
h
y
m
a
l
e
subject
s f
r
om
FM
GM
U, wh
o we
re 3
0
-
40 y
ears
ol
d,
h
a
d b
o
d
y
m
a
ss i
ndex <2
8 k
g
2
/m
2
, pl
at
el
et
s count
>1
0
0
,
0
0
0
/
m
m
3
, O bl
o
od t
y
pe,
no
rm
al pl
at
el
et agg
r
e
g
at
i
o
n
,
an
d wi
t
h
no
DM
, hy
pe
r
t
ensi
o
n
, an
d h
i
st
ory
of bl
oo
d di
so
r
d
ers
.
A
l
l
part
i
c
i
p
ant
s
were cap
abl
e
of gi
vi
n
g
i
n
f
o
rm
ed
consent.
2.1. Endotheli
a
l cells cultur
e
Medium
199 (M199)
was us
ing as
culture
m
e
dium
that
was prep
a
r
e
d
from
the powdere
d
M199
(Gi
b
co
) ad
de
d wi
t
h
2
g o
f
so
di
um
bi
carbo
n
a
t
e
and 2
g o
f
HEPE
S so
di
u
m
sal
t
s
. Growt
h
m
e
di
u
m
was M
1
9
9
su
pp
lem
e
n
t
ed
with
10
% fetal b
o
v
i
n
e
serum (FBS) (Caisso
n
),
10
0
IU/
m
L
o
f
p
e
n
i
cillin
and
10
0
µg/
m
L o
f
st
rept
om
y
c
i
n
(
S
i
g
m
a
), 0.
5%
f
u
n
g
i
z
o
n
e
(
G
i
b
co)
,
a
n
d
2
m
M
of
L-
gl
ut
am
i
n
e (Si
g
m
a
).
The um
bilical
cord buffe
r wa
s
prepa
r
ed
from
9.6
g of
Dul
b
ecco’s phos
phat
e buffered
s
a
line (PBS)
p
o
wd
er t
h
at d
i
sso
lv
ed
with
distilled
water in
to
1
000
m
L
i
n
to
tal vo
lu
m
e
. Th
e so
lu
tion was pu
t in
t
o
g
l
ass
b
o
ttles an
d
sterilized
with
auto
clav
e at
1
2
1
0
C, fo
r
15
m
i
n
u
t
es. After steril
izin
g
,
add
1
0
0
IU/m
L o
f
p
e
n
i
cillin
an
d 100
µg
/mL of st
rep
t
o
m
ycin
, th
at
ob
tain
ed fro
m
crystallin
e p
r
o
cai
n
e
p
e
n
i
cillin
-G
p
o
wd
er (M
eij
i
) and
st
rept
om
y
c
i
n
sul
f
at
e p
o
w
d
er
(M
ei
ji
) di
ss
ol
v
e
d wi
t
h
st
erilized
water for in
j
ection
.
Th
is b
u
f
fer so
lu
tion
was
kept
at
4
0
C.
Hum
a
n um
bilical vein endothelial cells (HUV
EC) c
u
lture
was esta
blishe
d according to t
h
e
t
echni
q
u
e d
e
sc
ri
bes by
Jaf
f
e
et al.
[1
0]
, Tanaka
(Sc
h
o
o
l
o
f
M
e
di
ci
ne, K
obe
Uni
v
ersi
t
y
) [1
1]
, an
d B
a
udi
n
et
al
.
[12] The
um
bi
lical cord t
a
ken
from
donors
was
placed in sterile bottle
contai
ned
buffe
r sol
u
tion (PBS-1%
Pen Stre
p) at 4
0
C un
til p
r
o
c
essed
in
the lab
o
ratory with
i
n
24
hou
rs
o
f
co
llectio
n
.
Aft
e
r th
e ou
tsid
e
o
f
the
um
bilical cord
was cleane
d
wi
th povi
don
e iod
i
n
e
, th
e l
u
m
e
n
of u
m
b
ilical
v
e
in
was id
en
ti
fied
. Cannu
la n
eed
le
was in
serted
i
n
to
th
e
v
e
in
an
d
k
e
p
t
tig
h
tly with
a cl
am
p. The
n
t
h
e
um
bi
l
i
cal
vei
n
wa
s fl
us
he
d wi
t
h
ri
n
g
e
r
lactate so
lu
tion
u
n
til it was
free
fro
m
remain
in
g b
l
o
o
d
.
Th
e
n
e
x
t
step
was
fillin
g
th
e u
m
b
ilical v
e
in
with
en
zym
e
so
lu
tio
n
to
d
i
sagg
regate EC. For th
at, warm
0
.
25
% tryp
sin
-
EDTA (Gib
co) was i
n
j
ected
in
t
o
the v
e
in
u
n
til it was d
i
sten
d
e
d
.
An
o
t
her
o
p
e
n
end
o
f
th
e co
rd
was
cla
m
p
e
d
.
Th
e
u
m
b
ilical co
rd was in
cu
b
a
ted fo
r
2
m
i
nut
es i
n
t
o
a beake
r
gl
ass fi
l
l
e
d wi
t
h
war
m
PB
S. Aft
e
r i
n
cu
bat
i
o
n peri
od
, t
h
e cl
am
p
was t
a
ken
of
f and t
h
e
enzym
e
solution
was c
o
llected int
o
a
sterile
bottle alrea
d
y filled with
1 cc
of FBS. The efflue
nt was re
pl
aced
i
n
t
o
a co
ni
cal
t
ube t
o
be ce
nt
r
i
fu
ged at
2
0
00
rpm
for
10 m
i
nut
es. T
h
e su
per
n
at
ant
was
rem
ove
d a
nd t
h
e
p
e
l
l
e
t
was wa
she
d
o
n
ce wi
t
h
M
1
9
9
. T
h
en t
h
e c
e
l
l
was resu
sp
ende
d
wi
t
h
g
r
owt
h
m
e
di
u
m
and i
noc
ul
at
ed o
n
a
g
e
latin
e-co
ated 6
0
mm
-d
ia
m
e
ter tissu
e cu
lture d
i
sh
(Iwak
i
). Th
e d
i
sh
was in
cub
a
ted
at 37
0
C
an
d
5% C
O
2
fo
r
24
h
o
u
r
s.
On t
h
e f
o
l
l
o
wi
ng
d
a
y
,
t
h
e gr
o
w
t
h
m
e
di
um
was
chan
ge
d aft
e
r t
h
e di
s
h
was
w
a
she
d
wi
t
h
PB
S-1
%
Pen St
rep t
o
re
m
ove re
d
blood cells. The
growt
h
m
e
dium was cha
nge
d eve
r
y 2
days until it reache
d
80%
con
f
l
u
e
n
ce.
T
h
e cel
l
s
were
o
b
s
erve
d
u
nde
r a
n
i
n
ve
rt
ed m
i
crosc
ope
(Ecl
i
p
s
e
, Ni
ko
n,
Ja
pa
n)
.
HUVEC pass
age 2 were
growth
on gel
a
tin-coate
d c
o
verslip afte
r reaching c
o
nfl
u
ence
we
re
characte
r
ized
by the prese
n
ce of von-W
illebrand factor
(vWF)
[13]-[
14]. Im
m
unocytoc
hem
i
st
ry was done for
cel
l
charact
eri
zat
i
on. M
o
n
o
l
a
y
e
r HU
VEC
on
gel
a
t
i
n
-c
oat
e
d co
ver
s
l
i
p
w
a
s fi
xe
d wi
t
h
m
e
t
h
anol
.
Ant
i
-v
WF
rabb
it p
o
l
yclon
a
l an
tib
od
y (1
:50
;
Novo
castra, Newcastle
, UK)
was use
d
as t
h
e pri
m
ary
ant
i
b
o
d
y
fo
r 1 h a
t
R
T
, f
o
l
l
o
we
d
by
t
r
eki
e
uni
v
e
rsal
l
i
nk
(St
a
r
r
Tre
k
, B
i
oca
r
e M
e
di
cal
, Net
h
erl
a
nds
) as t
h
e seco
nda
ry
an
t
i
body
fo
r
20 m
i
nut
es
at
R
T
. T
h
e c
o
versl
i
p
s we
re i
n
cu
bat
e
d
wi
t
h
Trek
A
v
i
d
i
n
h
o
r
se ra
d
d
i
s
h
pe
r
oxi
dase
(HR
P
)
l
a
bel
(St
a
r
r
Tre
k
) f
o
r 1
0
m
i
nut
es, and
fol
l
o
we
d b
y
bet
azoi
d
3
-
3
’
di
am
i
nobenzi
d
i
n
(DA
B
)
chro
m
o
g
e
n
(1
:50
;
Star
r
Trek
) fo
r 2 m
i
nut
es.
T
h
e cove
rsl
i
p
was
co
unt
e
r
st
ai
ne
d wi
t
h
M
a
y
e
r
’
s hem
a
t
oxy
l
i
n
(M
e
r
c
k
, Da
rm
st
adt
,
Germ
any
)
f
o
r
2 m
i
nut
es, a
n
d
t
h
e
n
un
de
rwe
n
t
d
e
hy
drat
i
o
n
seri
es
usi
n
g
et
han
o
l
a
n
d
xy
l
o
l
.
The
c
ove
rsl
i
p
was
fi
nal
l
y
m
ount
ed
on
a m
i
croscope
gl
ass
u
s
i
n
g
xy
l
e
ne-
b
ase
d
m
ount
i
n
g
m
e
di
um
(EZ-M
o
un
t
,
Sha
n
do
n,
T
h
erm
o
Scien
tific,
USA). Th
e cells
were ex
am
in
ed u
nde
r a
n
o
p
t
i
c
m
i
crosco
pe
(
O
l
y
m
pus,
Japa
n)
.
Evaluation Warning : The document was created with Spire.PDF for Python.
I
J
PH
S I
S
SN
:
225
2-8
8
0
6
Hi
g
h
Gl
uc
ose,
but
N
o
t
Test
ost
e
ro
ne,
Inc
r
eas
e
s Pl
at
el
et
Ag
g
r
eg
at
i
o
n
Medi
a
t
e
d
..
.. (
I
k
h
l
a
s Mu
h
a
m
m
a
d
J
e
ni
e)
20
7
2.
2.
Pl
atel
e
t
ri
ch pl
as
ma
Ven
o
u
s
bl
o
o
d
was c
o
l
l
ect
ed i
n
t
o
1/
1
0
v
o
l
u
m
e
of
3.
8% s
o
di
um
ci
t
r
at
e fr
om
t
h
e do
n
o
rs
.
The
w
hol
e
bl
o
od
was ce
nt
ri
f
uge
d at
9
00
rpm
for 1
5
m
i
nut
es. T
h
e supe
rnat
a
n
t
i
.
e. pl
at
el
et
ri
ch pl
asm
a
(PR
P
) was
col
l
ect
ed. T
h
e
rem
a
i
n
i
ng
bl
oo
d wa
s cent
r
i
f
u
g
ed at
1
5
0
0
rpm
for an
ot
her
15 m
i
nut
es t
o
get
pl
at
el
et
poo
r
plasm
a
(PPP).
The PR
P a
nd
PPP
were
ke
pt a
t
room
te
m
p
erature until platelet
aggre
g
ation test
was performe
d
with
in
3
ho
urs.
2.
3. T
r
e
a
t
men
t
Prim
ary HUVEC reache
d
80%
confl
u
ence
on the
6-9
th
day
.
The
HU
VEC
m
onol
ay
er was
t
r
y
p
si
ni
ze
d
wi
t
h
0.
25
% t
r
y
p
si
n
-
E
D
TA
an
d c
o
u
n
t
e
d
.
T
h
e
EC
we
re
pl
ated in a
gelatin-c
oated
24-well
microplate (Iwaki) at
2
x10
4
cells/well. After
24
hours the growt
h
medium
of
the seconda
r
y HUVEC
was
re
placed with
either NG
(5
.6
nM
)
or
H
G
M
1
99
g
r
o
w
t
h
m
e
di
u
m
(22
.
4nM
) wi
t
h
o
u
t
or
wi
t
h
T at
0
,
1,
10
, o
r
1
0
0
nM
. F
o
r t
h
e n
e
xt
2
4
ho
u
r
s, t
h
e t
r
ea
tm
ent
was rem
ove
d a
n
d t
h
e
m
onol
ay
er EC
were
wa
she
d
wi
t
h
PB
S
,
t
r
y
p
si
ni
ze
d
wi
t
h
0.
25
%
t
r
y
p
si
n
-
ED
TA
,
cent
r
i
f
u
g
ed
at
20
0
0
rpm
fo
r
5
m
i
nut
es, a
n
d r
e
sus
p
en
de
d
wi
t
h
50
0 µ
L
of
se
rum
free M
1
9
9
.
2.
4. Pl
atel
e
t
a
ggre
g
ati
o
n tes
t
Pl
at
el
et
agg
r
eg
at
i
on t
e
st
was
do
ne
wi
t
h
t
u
rb
i
d
im
et
ri
c
m
e
t
hode
usi
n
g a
n
a
g
g
r
e
gom
et
er m
achi
n
e
an
d
adenosi
n
e diphos
phate (ADP)
as
platelet
agonist, bo
t
h
were
f
r
om
Hel
e
na La
b
o
rat
o
r
y
. 5
µM
of
A
D
P
was
pre
p
are
d
by
di
l
u
t
i
ng
20 µM
of
ADP wi
t
h
0.
9
%
of sal
i
n
e wi
t
h
rat
i
o
1:
3
.
Li
g
h
t
t
r
ansm
i
t
t
a
nce
m
easurem
ent was
tak
e
n to
t
h
e
follo
wing
: an
emp
t
y cuv
e
tte, a
cu
v
e
tte
fille
d
with
3
00 µL
of aq
u
a
d
e
st, and
a cuv
e
tte filled
wit
h
45
0 µL o
f
PP
P. A
not
her
4 c
uvet
t
e
s we
re p
r
epa
r
ed
wi
t
h
a
m
a
gnet
i
c
st
ri
rrer i
n
si
de.
50
µL of e
n
d
o
t
h
el
i
a
l
cel
l
sus
p
ensi
on wa
s put int
o
each
cuvette, acc
ording to t
h
e de
si
gnate
d treatm
e
nt group. 50 µ
L
of PR
P was
adde
d
into the cuvette
to reach ratio 10
4
:
1
t
o
t
h
e EC
and i
n
c
uba
t
e
d fo
r 3 m
i
nu
t
e
s at
37
0
C
.
Fi
nal
l
y
, 50 µL o
f
5 µM
ADP was ad
d
e
d
in
t
o
th
e cuv
e
tte, an
d th
e
read
ing
o
f
ligh
t
transm
ittance for platelet
aggre
g
ation was
started.
Du
ri
n
g
1
0
m
i
nut
es
-rea
d
i
n
g,
vari
ab
els suc
h
as Max%,
TMax, slope,
lag and % area under curve were
recorde
d
. T
o
exam
ine the tre
a
tm
ent effect, Max% or m
a
xi
mal plate
l
et ag
gre
g
ation in pe
rcenta
ge was used as
th
e p
a
ram
e
ter.
2.
5. St
ati
s
ti
c
a
n
al
ysi
s
O
n
e way ANO
VA
w
a
s used
t
o
an
alyse th
e i
n
f
l
u
e
n
c
e of
T at
v
a
r
i
ou
s do
ses in
eith
er
N
G
or
HG
m
e
di
um
. Anal
y
s
i
s
of
va
ri
an
s was
use
d
t
o
anal
y
s
e
2x
4
fact
ori
a
l
desi
g
n
s.
p
val
u
e <
0
.
0
5
was
c
ons
i
d
ere
d
si
gni
fi
ca
nt
.
3.
R
E
SU
LTS AN
D ANA
LY
SIS
3.1. Endotheli
a
l cells cultur
e
Prim
ary EC cu
ltu
re was establish
e
d
fro
m
h
u
man
u
m
b
ilica
l
v
e
in
(h
u
m
an
u
m
b
ilical
v
e
in
en
do
th
elial
cells; HUVEC
)
usi
ng e
n
zym
a
tic digestion.
The e
n
do
thelia
l cell culture reach
ed confl
u
e
n
ce at 6-9
th
da
y
.
EC
cul
t
u
re
was i
d
e
n
t
i
f
i
e
d
by
m
o
rph
o
l
o
gi
cal
an
d
im
m
unol
o
g
i
cal
app
r
oac
h
. B
y
m
o
rp
h
o
l
o
gi
cal
i
d
ent
i
f
i
cat
i
o
n
usi
n
g
a p
h
ase c
ont
ra
st
m
i
crosco
pe,
EC
wer
e
o
b
se
r
v
ed
as m
ono
n
u
cl
eat
e an
d
po
l
y
gonal
e
p
i
t
h
el
i
a
l
cel
l
s
. A c
o
nfl
u
ent
m
onol
ay
er o
f
EC
sh
ow
ed
co
bbl
est
one
ap
pe
arance
as s
h
ow
n i
n
Fi
g
u
r
e
1.
Fig
u
re
1
.
Th
e
m
o
rp
ho
log
y
of a conflu
en
t mo
no
layer
o
f
end
o
t
h
e
lial cells
Evaluation Warning : The document was created with Spire.PDF for Python.
I
S
SN
:
2
252
-88
06
I
J
PH
S Vo
l. 4
,
N
o
. 3
,
Sep
t
emb
e
r
201
5
:
2
05
–
21
0
20
8
End
o
t
h
e
lial cells were
g
r
owth in
co
m
p
lete
med
i
u
m
as d
e
scrib
e
d
i
n
th
e Research
Met
h
od
.
Cells were
reache
d
c
o
nfluent after 6
day
s
and s
h
owe
d
t
h
e c
o
bble
stone
appeara
n
ce
of closely pac
k
e
d
m
ononucleate and
p
o
l
ygon
al endo
th
elial cells.
Cells w
e
r
e
ob
ser
v
ed
un
d
e
r
inv
e
r
t
ed
m
i
cr
o
s
co
p
e
w
ith 100
x
mag
i
n
i
f
i
catio
n
.
By im
m
unological identific
ation
using im
m
unocytoc
hem
i
stry
m
e
t
hod, the EC c
u
lture
positively
ex
pressed
vo
n W
illebran
d
facto
r
(vWF)
at
t
h
e cell m
e
m
b
ra
n
e
as shown in Figu
re 2.
Fig
u
re 2
.
Th
e p
o
s
itiv
e
exp
r
essio
n
of v
o
n
Wi
lleb
r
and
factor (vWF)
in
endoth
e
lial
cells
En
dot
hel
i
a
l
cel
l
s
fr
om
hum
an um
bi
l
i
cal
vei
n
(
H
UV
EC
)
p
a
ssage
2
were
gr
owt
h
on
a
g
e
l
a
t
i
n
-coat
e
d
cove
rsl
i
p
.
A
c
o
n
f
l
u
e
n
t
m
ono
l
a
y
e
r EC
wa
s
fi
xe
d a
n
d
immu
no
lab
e
lled
with
an
ti-vWF
po
lyclo
n
a
l an
tibo
d
y
as
descri
bed in the Resea
r
ch
Method.
The
vWF e
x
pressi
on was
detected
on the
cell m
e
m
b
rane
(brown).
Mayer’s h
e
m
a
to
x
y
lin
was
used
to
coun
terstain
th
e n
u
c
l
e
i (pu
r
ple
)
. C
e
lls were o
b
se
rve
d
u
n
d
er in
verte
d
m
i
crosco
pe wi
t
h
40
x
m
a
gni
fi
c
a
t
i
on.
3.
2. Pl
atel
e
t
a
ggre
g
ati
o
n tes
t
B
y
usi
n
g l
u
m
i
ni
sence
t
u
r
b
i
d
i
m
et
ri
c
m
e
t
hod
, m
a
xi
m
a
l platelet aggregation
was m
easured in PR
P
after in
cu
b
a
ted with
EC prev
i
o
u
s
ly treated
with
testo
s
terone
in NG or HG
m
e
diu
m
.
The
results
are presente
d
in
Tab
l
e 1.
Tabl
e
1. M
a
xi
m
a
l
pl
at
el
et
aggre
g
at
i
o
n m
e
di
at
ed by
e
n
dot
h
e
l
i
a
l
cel
l
s
t
r
eated
wi
t
h
t
e
st
ost
e
ro
ne a
n
d
gl
uc
ose
Testosterone level
in the
E
C
cultur
e
m
e
dium
(n
M)
Per
centage of
m
a
x
i
m
a
l platelet aggr
egation
m
e
diated by endothelial cells
fr
o
m
two differ
e
nt glucose content in
the cultur
e
m
e
diu
m
Norm
oglucose High
glucose
0
29.
3 ± 5.
8
46.
5 ± 10.
7
1
25.
0 ± 6.
8
43.
9 ± 1.
4
10
100
28.
5 ± 7.
0
29.
1 ± 9.
9
48.
1 ± 0
37.
4 ± 2.
3
Note
. M
a
xim
a
l p
l
at
ele
t
aggr
egat
i
on is
pres
ent
e
d a
s
mean ± SD from 2 indepe
nden
t
experimen
t
s. EC
= endoth
e
lial
cells. Normogluco
s
e
= 5.6 mM glucose in th
e EC
cultur
e
medium. High glu
c
ose
=
22.4 mM glucos
e in
th
e EC
cu
ltu
re medium.
As see
n
i
n
t
h
e
Tabl
e 1
,
pe
rce
n
t
a
ge
of m
a
xi
m
a
l
pl
atelet
ag
g
r
eg
ation
of PRP after in
cu
bated
with
EC
p
r
ev
iou
s
ly treated
with
NG med
i
u
m
was lo
wer th
an
of
PRP after in
cubated
with
EC prev
i
o
u
s
ly treated
with
HG m
e
d
i
u
m
, in
each
level of T i
n
th
e cu
lt
u
r
e m
e
d
i
u
m
. By o
n
e
-
w
ay
AN
OVA
an
alysis, ex
po
su
r
e
of T at
vari
ous
d
o
ses
t
o
EC
di
d
n
o
t
s
h
o
w
si
gni
fi
can
t
i
n
fl
ue
n
ces t
o
m
a
ximal platelet ag
greg
ation in
eith
er
NG
(
p
=
0.
14
4
)
o
r
HG m
e
di
um
(
p
= 0.91
6)
. By an
alysis o
f
v
a
r
i
an
s
f
o
r
2
x
4
f
actori
a
l design, t
h
ere
was a m
a
in effect
of
gl
uc
ose t
o
EC
t
o
m
a
xim
a
l
pl
at
el
et
aggre
g
at
i
on
(
p
= 0.004). Howe
ver, there was
no m
a
in effect of T t
o
EC to
maxim
a
l platelet aggre
g
ation
(
p
=
0
.
07
3)
.
M
o
re
o
v
er,
t
h
e
r
e was no
i
n
t
e
ract
i
on bet
w
ee
n T and
gl
uco
s
e
t
o
EC
t
o
maxim
a
l platelet aggre
g
ation
(
p
= 0.
69
).
Th
e aim
o
f
th
is stu
d
y
is to
investig
ate th
e in
flu
e
n
ce
o
f
T and
HG co
nd
ition
to
th
e
no
rm
al
fun
c
tion
of
EC in
prev
en
ti
n
g
p
l
atelet aggreg
ation
.
Th
is
in
v
itro
e
xpe
ri
ment is specifi
c to exam
ine the influe
nce
of T and
HG to
EC in
relatio
n
to
th
e early stag
e o
f
th
ro
m
b
o
s
is.
Howev
e
r, th
is
m
o
d
e
l is n
o
t
co
m
p
atib
le wit
h
real
co
nd
itio
n, in
wh
ich
h
y
p
e
rg
lyce
m
i
a o
r
T or the co
m
b
in
atio
n
co
nd
itio
n
will affect EC and
p
l
atelets at th
e same
ti
m
e
.
Evaluation Warning : The document was created with Spire.PDF for Python.
I
J
PH
S I
S
SN
:
225
2-8
8
0
6
Hi
g
h
Gl
uc
ose,
but
N
o
t
Test
ost
e
ro
ne,
Inc
r
eas
e
s Pl
at
el
et
Ag
g
r
eg
at
i
o
n
Medi
a
t
e
d
..
.. (
I
k
h
l
a
s Mu
h
a
m
m
a
d
J
e
ni
e)
20
9
Ro
senb
lu
m
et al.
[1
5]
ha
d
i
nve
st
i
g
at
ed t
h
e i
n
fl
uence
o
f
T to platelet
aggre
g
ation by im
planting
sub
c
ut
a
n
eo
usl
y
T or di
hy
d
r
ot
est
o
st
er
o
n
e (
DHT
) pel
l
e
t
s
i
n
m
i
ce, whi
c
h i
n
ject
e
d
wi
t
h
n
oxi
ou
s l
i
ght
/
dy
e
st
im
ul
us t
o
d
a
m
a
ge en
d
o
t
h
el
i
u
m
.
They
r
e
po
rt
ed
t
h
at
a
n
d
r
oge
n
pel
l
e
t
s
i
n
crea
sed
pl
at
el
et
aggre
g
a
t
i
on.
Ho
we
ver
,
w
h
e
n
i
s
ol
at
i
ng
pl
at
el
et
s of t
hose
m
i
ce, pl
at
el
e
t
s from
mice
treated with
T,
but not those treated
with
DHT, enh
a
n
c
ed
p
l
atelet reactiv
ity. Thu
s
, th
ey co
n
c
l
ude
d that the i
n
fl
uence
of
T in increasi
ng
platelet
aggre
g
ation wa
s not a
direct e
ffect to
p
l
atelets bu
t ind
i
rectly th
rou
g
h
EC
.
Act
u
al
l
y
, T d
o
e
s have a
di
rec
t
i
n
fl
ue
nce t
o
pl
at
el
et
s as had bee
n
i
n
vest
i
g
at
ed by
M
a
t
s
u
d
a
et al.
[16
]
in
rats. Th
ey repo
rted
th
at
rats treated
with
T
cypionate for 2
weeks ha
d a
n
increase
d
in platelets
throm
boxa
ne receptor
densit
y and platelet aggre
g
atio
n.
Ajayi &
Hal
u
skha [17]
found that throm
b
oxa
n
e
recept
o
r de
nsit
y in platelets
from
prostate cancer patie
nt
s unde
rwent surgi
cally and/
or medically castration is
was si
gni
fi
cant
l
y
l
o
wer
t
h
a
n
c
ont
rol
s
.
Ca
m
p
elo
et al.
[
18]
a
n
d C
u
t
i
n
i
et al.
[1
9]
of
fere
d a
n
ot
he
r a
p
p
r
o
ach
by
i
n
cu
bat
i
n
g
m
onol
ay
er
EC
fr
om
rat
aort
i
c
ri
n
g
s
wi
t
h
PR
P
an
d T at
vari
o
u
s
do
ses.
Aft
e
r
1-m
i
nut
e i
n
c
u
bat
i
o
n
,
PR
P
w
a
s rem
oved
f
r
o
m
t
h
e
pl
at
e an
d m
e
asure
d
pl
at
el
et
s agg
r
e
g
at
i
o
n
b
y
t
u
rbi
d
i
m
et
ri
c
m
e
t
hod
usi
n
g
5 µM
of
AD
P as pl
at
el
et
ago
n
i
s
t
.
They
f
o
un
d
t
h
at
i
n
cu
bat
i
n
g P
R
P wi
t
h
m
onol
ay
er EC
decre
a
sed
pl
at
el
et
agg
r
e
g
at
i
o
n
as c
o
m
p
ared t
o
PR
P t
h
a
t
was
not e
x
pos
ed to
EC.
However, they s
h
owed t
h
at the
pr
esence of T
de
press
e
d f
u
rt
he
r platelet aggre
g
ation.
What Cam
p
elo
et al.
[18
]
and Cu
tin
i
et al.
[
19]
had
re
po
rt
ed t
h
at
T
has a
n
t
i
-
ag
gre
g
at
or
y
act
i
on i
s
co
n
t
rary
t
o
what
Rose
n
b
lu
m
et al.
[1
5]
, M
a
t
s
uda
et al.
[16]
, an
d A
j
a
y
i
and Hal
u
s
k
ha [1
7]
had
pr
ove
d t
h
at
T ha
s pro
-
aggre
g
atory ac
tion. T
h
e
diffe
r
ence is t
h
e
re
su
l
t
o
f
t
h
e
des
i
gn
of
t
h
e e
x
p
e
ri
m
e
nt
. C
a
m
p
el
o
et al.
[
16]
an
d
Cu
tin
i
et al.
[1
9]
appl
i
e
d
T t
o
EC
and
pl
at
el
et
at the sa
m
e
tim
e, whereas
Rosenblum
et al.
[
15]
, M
a
t
s
u
d
a
et al
.
[1
6]
, an
d A
j
ay
i
and Hal
u
sk
ha
[1
7]
were m
o
re foc
u
s t
o
the expos
u
re of
T to
pl
atelet. Indeed, Khetawat
et al.
[20] showe
d
that androge
n
re
ceptors
we
re
d
e
t
ect
ed i
n
pl
at
el
et
. Di
ffe
red
wi
t
h
t
h
ose
pre
v
i
o
us e
xpe
ri
m
e
nt
s, i
n
th
is stud
y T
does n
e
ith
er increase no
r d
e
c
r
eas
e platelet aggre
g
ation through
EC.
It
has
be
en
est
a
bl
i
s
he
d t
h
at
di
abet
i
c
en
vi
r
o
n
m
ent
pr
om
ot
es en
dot
hel
i
a
l
pe
rt
ur
bat
i
o
n a
n
d
dy
sf
unct
i
o
n
.
HG condition in a
n
i
n
vitro study m
i
m
i
cs hyperglycem
ia found i
n
patients
with DM [1]. Th
ere
are
num
e
rous
evide
n
ces
for
the role of E
C
as a
break
for platelet aggre
g
ation; howev
e
r
,
o
n
ly
a few t
h
at re
po
rted th
e
in
flu
e
n
ce
of
HG co
nd
ition
to EC in relation to
p
l
atelet activ
atio
n. C
o
sen
tin
o
et al.
[2
1]
had
s
h
o
w
e
d
t
h
at
t
h
e
function of EC
in preve
n
ting platelet
aggre
g
ation is not optim
a
l in HG m
e
dium
because in this envi
ronment
EC
secret
e pr
ost
a
n
o
i
d
t
h
at
f
unct
i
o
ns as va
soc
onst
r
i
c
t
o
r a
nd
pr
o pl
at
el
et
aggre
g
at
i
o
n, i
.
e. t
h
r
o
m
boxa
ne A
2,
m
u
ch
m
o
re t
h
an pr
ost
a
noi
d t
h
at
funct
i
o
ns as
vaso
di
l
a
t
o
r
and
an
tip
latelet ag
greg
atio
n, i.e. p
r
ostacyclin
. It h
a
s
been est
a
bl
i
s
h
e
d al
so t
h
at
T
2
DM
pat
i
e
nt
s have e
x
a
gge
rat
e
d pl
at
el
et
react
i
v
i
t
y
[22]
-[
2
3
]
.
In a
g
r
eem
ent
wi
t
h
literatu
re, t
h
is
stu
d
y
sho
w
ed
t
h
at HG env
i
ron
m
en
t in
creases p
l
atelet agg
r
eg
atio
n throug
h
EC.
Shin
et al.
[2
4] an
d
C
h
eu
ng
et al.
[
2
5]
re
p
o
rte
d
that se
r
u
m
T leve
l was inve
rsely correlated wit
h
pl
asm
a
gl
ucos
e l
e
vel
,
w
h
e
r
e
a
s Her
n
a
d
ez-
M
i
jares
et al.
[2
6]
f
o
u
n
d
t
h
a
t
l
o
w T l
e
vel
was c
o
m
m
on am
ong
T2
DM
pat
i
e
nt
s
.
It
has bee
n
su
gge
st
ed t
h
at
l
o
w T l
e
vel
i
s
on
e of t
h
e co
nse
q
uence
s
of
DM
.
Thi
s
st
udy
sh
owe
d
th
at HG
d
o
e
s
no
t in
teract
with T in in
fl
u
e
n
c
in
g p
l
atelet aggreg
ation
t
h
rou
g
h
EC.
4.
CO
NCL
USI
O
N
Testosterone
does
not increa
s
e
platelet aggre
g
atio
n
thr
oug
h en
do
th
elial
cells,
bu
t h
i
gh
g
l
uco
s
e do
es.
ACKNOWLE
DGE
M
ENTS
We w
oul
d l
i
k
e t
o
t
h
an
k Di
rect
orat
e f
o
r Hi
g
h
er E
d
u
cat
i
on, M
i
ni
st
ry
of E
ducat
i
on
and C
u
l
t
u
re
,
R
e
pu
bl
i
c
o
f
In
do
nesi
a,
f
o
r
t
h
e R
e
searc
h
Gra
n
t
und
er “H
i
b
ah
D
e
sen
t
r
a
lisasi D
o
k
t
or
20
14
” Sch
e
m
e
.
REFERE
NC
ES
[1]
Popov, D., “Endothelial
cell d
y
sfunction in h
y
pergly
cemi
a: Phenoty
p
ic
change, intracellular sign
aling
m
odificat
ion, ult
r
astructur
a
l al
ter
a
tio
n
,
and poten
t
i
al c
lini
cal out
co
m
e
s
”
,
Int J Diabetes Mel
litus
, vo
l. 2, pp. 189-95
,
2010.
[2]
Schaffer
,
A., Bauersachs
,
J., “Endothelial d
y
s
f
unctio
n
,
impair
ed endogenous platel
et inhib
i
tion and platelet
act
ivat
ion in
di
a
b
etes
and
athero
s
c
leros
i
s
”
,
Curr Vasc
Pharm
, vol. 6
,
pp
. 52-60
, 2
008.
[3]
K
e
ll
y, D
.
M
., J
ones
,
T
.
H
., “
T
es
tos
t
er
one: a v
a
scular hormone in
hea
lth and
d
i
s
eas
e”
,
J Endo
crinol
, vo
l/issue
:
217(3), pp
. R47-
R71, 2013.
[4]
Torres
-
Es
ta
y,
V
.
, Carreno
, D
.
V
.
,
F
r
ans
i
s
c
o, I. F
.
S
., S
o
tom
a
y
o
r
,
P
., G
odo
y, A
.
S
.
, S
m
ith, G
.
J
., “
A
ndrogen rec
e
ptor
in
human endoth
e
lial cells”,
J En
docrinol
, vol. 22
4, pp
. R131-R13
7
, 2015
.
[5]
Liu,
P
.
Y.
, Death,
A.
K.,
Handelsman,
D. J., “Androgens and car
diovascular dis
ease”,
Endo
cr
in
e Re
v
, vol/issue:
24(3), pp
. 313-3
40, 2003
.
[6]
W
u
, F
.
C.
W
., v
on Eck
a
rds
t
ein
,
A
., “Androgens
and coron
a
r
y
arter
y
disease”,
En
docrin Rev
, vol.
24, pp. 183-217
,
2003.
Evaluation Warning : The document was created with Spire.PDF for Python.
I
S
SN
:
2
252
-88
06
I
J
PH
S Vo
l. 4
,
N
o
. 3
,
Sep
t
emb
e
r
201
5
:
2
05
–
21
0
21
0
[7]
J
ones
,
T. H
.
, S
a
ad, F
.
, “
T
he eff
ect of
testostero
ne on risk facto
r
s for,
and th
e
mediators of, th
e ath
e
rosclerotic
process”,
Athe
rosc
le
rosis
, vol. 20
7, pp
. 318-327
,
2009.
[8]
Broos, K., Fey
s
, H. B., De M
e
y
e
r, S. F., Vanhoorelbelke, K
., Deckm
y
n,
H., “Platelets at
work in primary
hem
o
s
t
as
is
”,
Blo
od Re
v
, vol. 25
,
pp. 155-167
, 20
11.
[9]
Buikema, J. H
.
, van Gilst, W.
H., “Endoth
e
lial function”, In
. Buikem
a, J.
H., “Endothelial D
y
sfunction in
Cardiovas
c
u
l
ar
Dis
eas
e: Rol
e
of P
h
arm
acolog
i
ca
l Int
e
rvent
i
o
n
s
”
,
Disse
rtation
, Groningen
:
Rijksuniversiteit
Groningen, 1995
.
[10]
Jaffe, E. A., Nachman, R. L.,
B
ecker
, C. G., Minick
, C. H., “C
ulture
of human
endothelial
cells derived from
hum
an um
bilica
l
veins”
,
J C
lin In
ves
t
, vol. 52
, pp
. 2745-2756, 197
3.
[11]
Tanak
a
, A., “Primar
y
cultu
r
e
of
HUVEC”, A han
dout, Schoo
l of
Medicin
e
, Kobe
University
, 1994
.
[12]
Baudin, B
.
, Bru
n
eel
, A., Bossel
u
t, N., Vaubour
dolle, M.,
“A protocol for
isolati
on and cu
ltur
e
o
f
hum
an um
bilical
vein endothelial cells”,
Nat Proto
c
, vo
l/issue: 2(3)
, pp
. 481-485
, 2
007.
[13]
Lip, G. Y. H.,
Blann, A., “Von Willebrand f
act
or: a
m
a
rker of
endothelial d
y
sf
unction in v
a
scular disorders
?
”,
Cardiov
a
sc
Re
s
,
vol. 34
, pp
. 255–
265, 1997
.
[14]
Mannucci
, P. M., “von Willebrand F
actor: A Marker of Endoth
e
li
al Dam
a
ge?
”
,
Arte
riosc
l
e
r
Thromb Vasc
Biol
,
vol. 18
, pp
. 1359
-1362, 1998
.
[15]
Rosenblum
, W
.
I., El
-Sabban,
F., Nelson,
G. H
., Allison,
T. B.
, “
E
ffec
t
s in m
i
ce of testosterone
a
n
d
dih
y
dro
t
estoster
one on platelet
aggregati
on in injured ar
terio
l
es
and ex vivo”,
Thromb R
e
s
, vol. 45, pp. 719-72
8,
1987.
[16]
M
a
ts
uda, K
., R
u
ff, A
., M
o
rinel
li, T
.
A
., M
a
thu
r
, R. S
., M
a
thur
, R. S
., H
a
lus
h
k
a
, P
.
V
., “
T
es
to
s
t
erone incr
eas
e
s
throm
boxane A2 recep
tor densit
y and respons
iven
ess in rat aor
t
as
and pla
t
el
ets”
,
A
m
J Phys
iol
, vol. 267, pp. H887-
H893, 1994.
[17]
Ajay
i, A
.
A.
L.,
Halushka, P. V
.
,
“Castration r
e
d
u
ces platelet
thr
o
mboxane A
2
receptor d
e
nsity
a
nd aggreg
ability”,
Q J Med
, vol. 98
, pp
. 349-356
, 2
005.
[18]
Campelo, A. E., Cutini, P. H
.,
Massheimer, V. L., “Testostero
n
e modulat
es platelet aggr
egatio
n and endothelial
cell growth
thro
ugh nitr
ic ox
ide
pathway
”
,
J End
o
cr
inol
, vol. 213
, pp
. 77-87
, 201
2.
[19]
Cutini,
P. H.
, Ca
m
p
elo, A.
E.
,
Agrielo
,
E., Sando
val, M
.
J., Rauschemberg
er, M
.
B., M
a
s
s
h
eim
e
r
,
V
.
L.
, “
T
he
rol
e
of sex steroids on cellu
lar ev
ents
involved in vas
c
ular disease”,
J Steroid Bioch
e
m Mol Biol
, vol/issue: 132(3-5),
pp. 322-330
, 20
12.
[20]
K
h
etaw
at
, G
.
,
F
a
rada
y,
N
.
, N
eal
en,
M
.
L.
,
V
i
aja
y
a
n
,
K
.
V
., Bo
lton
,
E
.
,
N
oga, S
.
J
.
,
Br
a
y
, P
.
F
.
, “
H
um
an
m
e
gakar
y
oc
yt
es
and platel
ets
conta
i
n the es
tr
ogen recep
tor beta and andro
g
en recep
tor (AR): Tes
t
os
tero
n
e
regulates AR
ex
pression”,
Blood
, vol. 7
,
pp
. 2289
-2296, 2000
.
[21]
Cosentino, F., Eto, M., D
e
Pao
lis, P., van
der
Lo
o, B., B
achsch
imd, M., Ul
lrich, V., Kouroedov, A., Gatti, C. D.,
J
o
ch, H
., V
o
lp
e,
M
., Lus
c
he
r, T
.
F
., “
H
igh gluco
s
e caus
e
s upregu
l
ation of
cy
cloo
x
y
gen
a
se-2 and
alters prostanoid
profile
in human endothelial cells
: role of pr
o
t
ein
kinase C and r
e
activ
e ox
y
g
en species”,
Circula
tio
n
, vol. 107
, pp
.
1017-1023, 200
3.
[22]
V
i
nik, A
.
I.
, Erb
a
s
,
T.
, P
a
rk, T
.
S
., N
o
lan, R.
, Pi
tteng
er, G. L
., “
P
latel
e
t d
y
sfunction in ty
pe 2 diabetes”,
Diabetes
Care
, vol/issue:
24(8), pp
. 1476-
1485, 2001
.
[23]
S
c
hneider
, D. J
.
, “
F
actors
con
t
ri
buting to
incr
ea
s
e
d plat
el
et r
e
a
c
tivit
y
in p
e
ople
with diab
et
es
”,
Diabe
te
s Care,
vol/issue: 32(4), pp.
525-527
,
20
09.
[24]
Shin, J. Y., Par
k
, E. K
., Park
, B. J., “High-n
o
rm
al glucose levels in non-d
i
abet
ic and pr
e-
diabetic men ar
e
as
s
o
ciat
ed
with
decer
as
ed tes
t
os
t
e
rone lev
e
ls
”,
K
o
rean J
Fam M
e
d
, vol. 33
, pp
. 15
2-156, 2012
.
[25]
Cheung, K
.
K.
T., Luk
,
A. O.
Y., So, W. Y.,
Ma, R.
C. W.,
Kong, A. P. S., Chow
, F. C. C., Ch
an, J. C
.
N
.,
“
T
estosterone l
e
vel in m
e
n wit
h
t
y
p
e
2 di
abe
t
es m
e
llitu
s and
rela
ted m
e
t
a
bo
lic
effe
cts: A r
e
view of
curren
t
eviden
ce”
,
J Dia
b
Inves
t
,
vo
l. 6,
pp. 112-123
, 20
15.
[26]
Hernandez-Mijares, A., Garcia-
M
alpart
ida,
K.,
Sola-Izqui
erdo,
E.
, “
T
estost
ero
n
e lev
e
ls i
n
m
a
les with
t
y
pe
2
diabetes and
their relationship w
ith cardiov
a
sc
ular risk f
actors
and card
i
ovascular disease”,
J S
e
x
Med
, vol. 7, pp.
1954-1964, 201
0.
Evaluation Warning : The document was created with Spire.PDF for Python.