Nutrition Research and Practice (2008), 2(2), 74-79
ⓒ2008 The Korean Nutrition Society and the Korean Society of Community Nutrition
Zinc deficiency decreased cell viability both in endothelial EA.hy926 cells and
mouse aortic culture ex vivo and its implication for anti-atherosclerosis*
Young-Eun Cho1, Jee-Eun Choi1, Md. Jahangir Alam1, Man-Hyo Lee2, Ho-Yong Sohn1, John H. Beattie3 and
1Department of Food Science and Nutrition, Andong National University, Gyungpook 760-749, Korea
2Gyeongbuk Institute for BioIndustry, Andong, Gyungpook, Korea
3Division of Vascular Health, Rowett Research Institute, Aberdeen, UK
Received May 16, 2008; Revised June 11, 2008; Accepted June 18, 2008
Zinc plays a protective role in anti-atherosclerosis but the clear mechanism has not been proposed yet. In the present study, we evaluated whether
zinc modulates atherosclerotic markers, VACM-1 and ICAM-1 and cell viability both in endothelial cells in vitro and mouse aortic cell viability
ex vivo. In study 1, as in vitro model, endothelial EA.hy926 cells were treated with TNFα for 5 hours for inducing oxidative stress, and then
treated with Zn-adequacy (15 μM Zn) or Zn-deficiency (0 μM Zn) for 6 hours. Pro-atherosclerosis factors, VCAM-1 and ICAM-1 mRNA expression
and cell viability was measured. In study 2, as ex vivo model, mouse aorta ring was used. Mourse aorta was removed and cut in ring then, cultured
in a 96-well plate. Aortic ring was treated with various TNFα (0-30 mg/ml) and intracellular zinc chelator, N, N, N′, N′, -tetrakis (2-pyridylmethyl)
ethylenediamine (TPEN, 0-30 μM) for cellular zinc depletion for 2 days and then cell viability was measured. The results showed that in in vitro
study, Zn-adequate group induced more VCAM-1 & ICAM-1 mRNA expression than Zn-deficient group during 6-hour zinc treatment post-5 hour
TNF-α treatment, unexpectedly. These results might be cautiously interpreted that zinc would biologically induce the early expression of anti-oxidative
stress through the increased adhesion molecule expression for reducing atherosclerotic action, particularly under the present 6-hour zinc treatment.
In ex vivo, mouse aortic ring cell viability was decreased as TNF-α and TPEN levels increased, which suggests that mouse aortic blood vessel
cell viability was decreased, when oxidative stress increases and cellular zinc level decreases. Taken together, it can be suggested that zinc may
have a protective role in anti-atherosclerosis by cell viability in endothelial cells and aorta tissue. Further study is needed to clarify how pro-atherosclerosis
molecule expression is modulated by zinc.
Key Words: Zn-deficiency, endothelial cells, EA.hy926 cell, mouse aorta, atherosclerosis
Atherosclerosis is a chronic inflammatory disease of arterial
blood vessels and the development of atherosclerosis is
influenced by genetic, lifestyle and nutritional factors (Beattie
& Kwun, 2004; Henriksen et al., 2008). At present time,
atherosclerosis and its complications remain a major cause of
morbidity and mortality worldwide. Cardiovascular and cerebro-
vascular diseases cause over 15 million deaths every year - one
third of the global total and coronary heart disease and stroke
accounts for 7.2 million and 4.6 million, respectively (Kharbanda
& MacAllister, 2005).
Initiation of an atherosclerotic lesion involves an endothelial
cell pro-inflammatory state by the oxidative stress that recruits
leukocytes and promotes their movement across the endothelium.
These processes require endothelial expression of adhesion
molecules such as vascular cell adhesion molecule-1 (VCAM-1),
intercellular adhesion molecule-1 (ICAM-1) and E-selectin.
Proinflammatory cytokines, such as TNF-a, IL-1b, IL-4,
accelerate atherosclerosis by inducing adhesion molecules of
endothelium, such as intercellular cell adhesion molecule-1
(ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)
(Zheng et al., 2008). ICAM-1 and VACM-1 are two members
of the Ig-like supergene family of adhesion molecules that are
normally expressed by endothelial cells (Papagianni et al., 2003)
and also in the aortic cells (Desai et al., 2007).
Several studies indicate that zinc is vital to vascular endothelial
cell integrity and for preventing severe impairment of the
endothelial barrier function (Beattie & Kwun, 2004; Clair et al.,
1995; Hennig et al., 1992; McClain et al.,1995), since the
endothelial dysfunction is considered as a surrogate marker of
vascular pathology leading to atherosclerosis (Minqin et al.,
*This work was supported by Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (Ministry of Science and Technology,
MOST; grant No. F01-2006-000-10172-0) and in part Korea Research Foundation (KRF, grant No. KRF-2007-313-C00810)
§Corresponding Author: In-Sook Kwun, Tel. 82-54-820-5917, Fax. 82-54-820-6188, Email. firstname.lastname@example.org
Young-Eun Cho et al.
Fig. 1. The endothelial EA.hy926 cell morphology by Zn treatment at 6 hours.
The cells were exposed to TNFα (10 ng/ml) for 5 hours and after then, the cells
were treated with TPEN (5 or 10 µM) and either Zn-deficient (0 μM Zn as ZnCl2,
Zn-) or Zn adequate (15 μM Zn as ZnCl2, Zn+) medium for 6 hours. ZnCl2 was
used for making 10 mM Zn stock solution. A: Zn chelator 10 µM TPEN with Zn-
(0 μM) or Zn+ (15 μM). B: Zn chelator 5 µM TPEN with Zn- (0 μM) or Zn+ (15
2007). It is also reported that zinc is inversely correlated with
the atherosclerotic lesion formation (Ren et al., 2003), which may
be through the anti-atherogenic properties by inhibiting activation
of oxidative stress-responsive transcription factors that are
activated during an inflammatory response in atherosclerosis
(Watt et al., 2006). Therefore, zinc can slow down the
progression of atherosclerosis, perhaps through its anti-infla-
mmatory properties and its ability to suppress the proliferation
of the smooth muscle cells adding to intimal thickening (Berger
et al., 2004; Reiterer et al., 2004). Moreover, recently from the
pooled microarray data, it is reported that genes involved in
cellular zinc homeostatis are modulated in atherosclerotic patients
under the state of treatment for decreasing cholesterol synthesis,
which implies zinc involvement in atherosclerosis incidence
(Costarelli et al., 2008).
In the present study, we evaluated whether zinc deficiency
decreased the viability of endothelial cells and mouse aortic ring
cell ex vivo model. Also, gene expression of VCAM-1 and
ICAM-1 in endothelial cells was assessed to evaluate zinc
modulation of CAMs. The study results could imply the potential
role of zinc for preventing atherosclerosis.
Materials and Methods
Chemicals were obtained from Sigma (St Louis, MO), unless
otherwise noted. Cell culture reagents (DMEM, penicillin, HAT,
streptomycin and fetal bovine serum) were obtained from Gibco
Laboratories (Grand Island, NY, USA). All consumables and
plastic wares were used with trace element free products or were
soaked in 10% nitric acid solution to prevent zinc and other trace
Study 1: In vitro study (EA.hy926 cell line)
Cell culture and zinc treatment
The human endothelial cell line (EA.hy926; ATCC: CRL-
2922) was originally derived from human umbilical vein and
showed vascular endothelial cell characteristics. EA.hy926 cells
were cultured in DMEM culture medium supplemented with 10%
FBS, 2% HAT medium supplement (the final concentration of
hypoxanthine, aminopterin, and thymidine was 100, 0.4 and 16
mM, respectively) and 1% penicillin/streptomycin in a humi-
dified atmosphere of 5% CO2 at 37℃. The cells were treated
just post-confluence because the differentiation marker for
endothelial cell, von willebrand factor, was optimally expressed
just post-confluence in this EA.hy926 cell line (Fig. 1 A-B).
Cellular zinc was depleted using intracellular zinc chelator, N,
N, N′, N′, -tetrakis (2-pyridylmethyl) ethylenediamine (TPEN).
Just post-confluence, the cells were treated with either Zn- (0
μM as ZnCl2) or Zn+ (15 μM as ZnCl2) with TPEN (5 or 10
µM) for 6 hours. After then, the cells were exposed to TNFα
(10 ng/ml) for 5 hours for inducing cellular oxidative stress.
Stock ZnCl2 solution (10 mM) was prepared in appropriate
concentration. The media were changed in every 2-3 days.
The cells were incubated with zinc, either in Zn- (0 µM) or
Zn+ (15 µM) with two different levels of intracellular zinc
chelator TPEN (5 or 10 µM TPEN) for 6 hours and then, were
treated with TNF-α (10 ng/ml) for another 5 hours. The
morphological features of EA.hy926 cell line were assessed by
inverted light microscope (Leica DMIL, Leica Microsystems,
WetZlar, Germany) to observe the bulk morphology for apoptosis
for both at 5 μM and 10 μM TPEN treatment.
Trypan blue test
The cells were incubated with zinc either in Zn- (0 µM Zn
+ 10 µM TPEN) or Zn+( 15µM Zn + 10 µM TPEN) for 6 hours
and then, were treated with TNF-α (10 ng/ml) for another 5 hours.
The cell viability of EA.hy926 cell was determined by the trypan
blue exclusion assay, in which if the dead cell membrane is
leaking and then dead cells uptake trypan blue dye while viable
RT- PCR for von Willebrand factor and cellular adhesion
molecules (VCAM-1 and ICAM-1)
mRNA expression of von Willebrand factor as endothelial cell
differentiation and vascular marker, and cellular adhesion
Zinc deficiency decreases cell viability in endothelial cells and in mouse aorta ex vivo
Table 1. Primer base sequence for target and housekeeping genes
Genes Base sequences
von Willebrand factor
molecules (VCAM-1 and ICAM-1) was measured by RT-PCR.
For RNA extraction from EA.hy926 cells, Trizol reagent
(Invitrogen, USA) was used as commercial instruction. For the
synthesis of first-strand cDNA, 100 ng of RNA from each sample
were reverse transcribed using 20 U of AMV reverse transcriptase
and oligo-p (dT) 1X random primers. The resulting cDNAs were
PCR-amplified by using a mixture of the corresponding sense
(forward) and antisense (reverse) primers (Table 1). Primers for
target and housekeeping genes were obtained from Bioneer
Corporation (Daedeok-gu, Daejeon 306-220, Korea). The PCR
conditions were 95℃ for 2 min and then 36 cycles at 95℃ for
30s, 55℃ for 45s and 70℃ for 1 min and a final extension at
72℃ for 5 min. The PCR products were separated on 1.8%
Study 2: Ex vivo study (mouse aortic ring culture study)
Mouse aortic ring culture
Mouse aorta from C57BL/6 strain was removed and all adipose
tissues were removed. The aorta was dissected in three major
parts through the entire aorta for random sampling and then, the
aorta was dissected 0.5 mm in thickness for the aortic ring
samples to culture in a culture dish, as ex vivo. Mouse aortic
ring was cultured in a 96-well plate as one ring per well in
DMEM culture medium supplemented with 30% FBS and 1%
penicillin/streptomycin in a humidified atmosphere of 5% CO2
at 37℃. The aortic ring culture was carefully examined overnight
and cultured for another two to three days, until the aortic ring
culture being confirmed viable. After confirming the aortic ring
being viable, then MTT assay was done for evaluating aortic
ring cell viability.
MTT assay for viability of mouse aortic ring cells
Mouse aortic ring cell viability was determined by the MTT
assay on the metabolic reduction of 3-[4, 5-dimethylthiazol-
2-y]-2, 5-diphenyltetrazolium bromide (MTT). Aortic ring was
plated in a 96-well plate and maintained in growth media for
2-3 days at 5% CO2 at 37℃. After then, the aortic ring was
treated with TNFα with the range of 0-30 ng/ml to evaluate aortic
cell viability by oxidative stress or TPEN with the range of 0-30
mM to evaluate if cellular zinc depletion by TPEN decreased
aortic ring cell viability for 2 days. Ten micorliter of MTT
solution was added in each well and incubated at 37℃ for 3
hours to allow for the formation of formazan crystals. After
formation of formazan crystals, the MTT medium was then
aspirated and replaced with solubilization solution (DMSO) for
dissolving the formazan crystals. The plates were read on Micro
Elisa reader (Asys Hitee, Expert 96, Asys Co., Austria) at 570nm.
In vitro study
The cells were shown to be less viable morphologically in zinc-
and the viability was much less in higher Zinc chelator treatment
group (10 μM TPEN), where less free zinc was available by
cells than in lower Zinc chelator group (5 μM TPEN) (Fig. 1).
The cells in Zn- showed fewer adherents to the cell dish and
visually apoptotic. zinc deficiency decreased endothelial cell
viability even within 6 hours, and the less viable morphology
was observed in higher zinc chelator level at 10 μM TPEN.
Trypan blue test
The cell viability was decreased by zinc deficiency (0 μM Zn)
for 6 hours. Seventy five percent cell viability was observed at
the Zn- (0 µM Zn + 10 µM TPEN), while Zn- (15 µM Zn +
10 µM TPEN) showed 94% of cell viability of 6 hours zinc
treatment, followed by 10 ng/ml TNFα treatment (data not
mRNA expression for von Willebrand factor, VCAM-1 & ICAM-1
To confirm if this particular cell line was appropriate for testing
endothelial characteristics, von Willebrand factor was assessed,
since von Willebrand factor is related to the formation of clots
and also can be a characteristic of endotherial cells. von
Willebrand factor on endothelial cells was expressed from the
just pre-confluence and was maximally from the post-confluence
2 days and maintained up to 6 days (Fig. 2 A). Therefore, we
decided to treat zinc just post-confluence on 2 days on this cell
model. The cells were treated for 6 hours with the level of Zn
– (1µM) and Zn+ (15 µM), and after then, the cells were exposed
with TNFα for 5 hours. mRNA expression of adhesion molecules,
VCAM-1 and ICAM-1, were measured by RT-PCR. The mRNA
expression of VCAM-1 and ICAM-1 mRNA was higher in Zn+
than Zn-, unexpectedly, under this experimental condition. Even
ICAM-1 mRNA expression showed a bit less discrepancy
between Zn- and Zn+ (Fig. 2 B, data are presented as the repres-
entative of three replicates). For the atherosclerotic incidence,
once inflammatory stimuli signal to the vascular endothelium,
which then in turn mediates leukocyte recruitment and adherence
to the endothelium at the early stages of vascular inflammation,
Young-Eun Cho et al.
Fig. 2. (A) Confirmation of von Willebrand factor gene expression in EA.hy926
cells. Zero day means when the cells were at confluence. (B) Representative gene
expression image of VCAM-1 and ICAM-1 of 6 hour zinc treatment in EA.hy
926 cells. The cells were exposed to TNFα (10 ng/ml) for 5 hours and after then,
the cells were treated with TPEN (5 or 10 µM) and either Zn- (0 μM) or Zn+ (15
μM) for 6 hours. The experiment was done in triplicate.
Table 2. MTT viability assay under different TNFα concentration for mouse
aorta cells at 2 days treatment
100.00 ± 18.2a
80.8 ± 27.3ab
78.4 ± 14.4ab
64.7 ± 35.2b
5.00 ± 5.7c
Data represent mean ± SEM (n=8), Tukey’s test, ANOVA at p < 0.05
Table 3. MTT viability assay under different TPEN concentration for mouse
aorta cells at 2 days treatment
100.0 ± 48.2a
124.4 ± 4.5a
56.6 ± 5.7b
1.1 ± 3.6c
-1.7 ± -2.3c
Data represent mean ± SEM (n=8), Tukey’s test, ANOVA at p < 0.05
ultimately leading to the progression of numerous vascular
diseases. VCAM-1 and ICAM-1 are responsible for the process
of formation of endothelium adherence.
Ex vivo study
MTT viability for mouse aorta by TNFα & TPEN
The functional integrity of the aortic ring, under zinc
deficiency, was assessed using the MTT viability assay for mouse
aortic ring culture. At first, we evaluated if cell viability affected
in this particular ex vivo mouse aortic ring culture sample which
was treated by TNFα, the inducer of oxidative stress. The
viability of mouse aorta cells was inversely proportional to the
TNFα concentration (0-30 ng/mL, Table 2), which confirmed the
TNFα-induced oxidative stress, and again which decreased mouse
aortic ring viability. On considering the 0 ng/mL of TNFα is
100 % cell viability, 10 ng/ml TNFα treatment showed marginal
viability level (78.4%), and then we decided to treat with this
level for consecutive zinc chelator experiment for mouse aortic
ring viability by cellular zinc deficiency. Then, we evaluated
whether less cellular zinc availability could decrease mouse aortic
ring viability. Cellular zinc depletion was induced by increasing
the addition of intracellular zinc chelator, TPEN (0-30 μM). The
aortic ring cell viability showed that as the added TPEN level
increased, which means less cellular zinc is available, the aortic
ring cell viability was decreased as shown in Table 3. The results
showed that as cellular zinc was less available, then aortic cell
The present study evaluated the potential role of zinc on
vascular disease induced by oxidative stress, in that whether
cellular zinc deficiency decreased the viability of both the
endothelial cell in EA.hy926 cell line and the mouse aortic cells
ex vivo model. Also it was examined whether cellular zinc
deficiency modulated the mRNA expression of VCAM-1 and
ICAM-1 in endothelial cells. To confirm beforehand if this
particular endothelial cell line, EA.hy926 cells, shows the
appropriate condition for vascular and inflammatory morphology,
von Willebrand factor (vWF) gene which is considered as the
endothelial cell differentiation was measured and confirmed vWF
gene expression in the cells under the present study condition.
vWF mRNA was expressed from 0 to 6 days of EA.hy 926 cell
culture after TNFα treatment. vWF is normally synthesized by
the endothelial cells and stored in the Weibel-Palade bodies and
it is secreted on endothelial activation (Collins et al., 1993).
Normally, the increased plasma vWF levels are associated with
cardiovascular disease (Theilmeier et al., 2008).
In the present study, the results showed that zinc could protect
endothelial cell viability both in endothelial in vitro and mouse
aortic ex vivo model under TNFα-induced oxidative stress
condition, which causes cardiovascular abnormality such as
atherosclerosis. TNFα induce the inflammatory signal for
increasing the oxidative stress and thus increase the cell apoptosis
through stimulating the oxidative stress in endothelial cells (Ling
et al., 2007). Ex vivo study showed that as more cellular zinc
chelator TPEN was added, which means less cellular zinc is
available, then the mouse aortic ring culture viability was
decreased under TNFα treatment (Table 3). In vitro study results
also showed as being consistent with the results of ex vivo, in
which zinc deficiency (Zn-) followed by TNFα treatment showed
more apoptotic and lower cell viability than zinc adequacy (Zn+)
(Fig. 1 and trypan blue test results).
In fact, it is reported that zinc deficiency induces cell death
in various cellular models (Clegg et al., 2005), however, not
much confirmed in endothelial cell models and nor in mouse
aortic ring culture model, ex vivo. The endothelial cell model
from porcine pulmonary arteries showed lower cell viability
Zinc deficiency decreases cell viability in endothelial cells and in mouse aorta ex vivo
under zinc deficiency induced by TPEN zinc chelator (Hennig
et al., 1999; Meerarani et al., 2000). Therefore, the findings in
the current study confirmed that zinc could protect cell death
in human origin endothelial EA.hy926 cells, which has not been
reported before, in addition to mouse aortic ring culture
condition. The preventive role of zinc for the decreased cell
viability in endothelial cells and mouse aortic tissue culture in
the current study results can be interpreted that zinc can increase
endothelial cell viability under oxidative stress, which can be
protective for atherosclerosis in the endothelium of blood vessels.
In our in vitro model, Zn- showed low cell viability than Zn+
in human origin endothelial EA.hy926 cells at 6-hour zinc
treatment prior to TNFα treatment, which causes intracellular
oxidative signal. Under TNFα exposure, the high cell viability
in Zn+ might result from the anti-oxidative property of zinc, since
zinc has an antioxidant role as a component of systemic
antioxidant enzyme such as Cu/Zn-SOD (Formigari et al., 2007).
The intracellular oxidative signals may provide a molecular
mechanism linking the oxidative signal which is one of risk
factors for the early pathogenesis of atherosclerosis through the
expression of some vascular inflammatory genes, such as cell
adhesion molecules VCAM-1 and ICAM-1 (Alexander et al.,
1995; Kunsch & Medford, 1999). It is reported that the cell
adhesion molecules VCAM-1 and ICAM-1 are expressed in
endothelial cells by oxidative stress, such as TNFa and other
inflammatory molecules (Choi et al., 2004; Reiterer et al., 2004).
In the present study, mRNA expression of VCAM-1 and ICAM-1
under zinc treatment in EA.hy926 cells was evaluated and
unexpectedly, VCAM-1 and ICAM-1 mRNA expression was
highly expressed in Zn+ under the current experimental condition
of 6-hour zinc treatment (1 μM or 15 μM) prior to TNFα
exposure (10 ng/ml). The potential explanation of the current
contradicting results for VCAM-1 and ICAM-1 mRNA
expression might be due to shorter zinc treatment time period
(6 hours with 1 or 15 μM Zn) and stronger TNFα exposure
condition (10 ng/ml for 5 hours), compared to longer Zinc
treatment (20 μM for 24 hours) and weaker TNFα exposure (10
ng/L for 2 hours) (Reiterer et al., 2004). Further study for
VACM-1 and ICAM-1 expression under optimal experimental
condition would be needed for getting final conclusion.
Normally, more endothelial adhesion molecules, VCAM-1 and
ICAM-1, induce more oxidative stress, which may cause the
progression of atherosclerosis. Even under the current particular
experimental condition, a potential explanation for higher mRNA
expression VCAM-1 and ICAM-1 in Zn+ would be suggested:
One possibility would be that the VCAM-1 and ICAM-1 were
up-regulators of atherosclerosis, which means these were
expressed normally before the progression of atherosclerosis. In
our study, Zn+ showed more VCAM-1 and ICAM-1 expression
which biologically induces more expression of anti-oxidative
enzymes. Recent study showed that the antioxidant defense
system against lipid peroxidation was increased, which can
propose to retard the development of atherosclerosis. Also, the
increase in Cu/Zn-superoxide dismutase (Cu/Zn-SOD) activity
may reduce vascular cell-mediated oxidation of low-density
lipoprotein in mouse aorta (Yang et al., 2004).
Taken all together, zinc adequate can protect endothelial
damage which is induced by oxidative stress through increasing
endothelial cell viability and aortic ring culture cell viability.
Also, zinc modulates cellular adhesion molecules (VDAM-1 and
ICAM-1) mRNA expression in endothelial cells. These findings
are cautiously interpreted that zinc can be a factor for preventing
atherosclerosis incidence. Further investigation will undertake for
clarifying the mechanism by zinc for anti-atherosclerosis.
Alexander RW (1995). Hypertension and the pathogenesis of
atherosclerosis: oxidative stress and the mediation of arterial
inflammatory response: a new perspective. Hypertension 25:155-
Beattie JH & Kwun IS (2004). Is zinc deficiency a risk factor for
atherosclerosis? Br J Nutr 91:177-181.
Berger M, Rubinraut E, Barshack I, Roth A, Keren G & George J
(2004). Zinc reduces intimal hyperplasia in the rat carotid injury
model. Atherosclerosis 175:229-234.
Choi JS, Choi YJ, Park SH, Kang JS & Kang YH (2004). Flavones
mitigate tumor necrosis factor-a-induced adhesion molecular
upregulation in cultured human endothelial cells: role of nuclear
factor-kB. J Nutr 134:10103-1019.
Clair J, Talwalkar R, McClainC J & Hennig B (1995). Selective
removal of zinc from cell culture media. Journal of Trace
Elements in Experimental Medicine 7:143-151.
Clegg MS, Hanna LA, Niles BJ, Momma TY & Keen CL (2005).
Zinc deficiency-induced cell death. IUBMB Life 57:661-669.
Collins PW, Macey MG, Cahill MR & Newland AC (1993). Von
Willebrand factor release and P-selectin expression is stimulated
by thrombin and trypsin but not IL-1 in cultured human
endothelial cells. Thromb Haemost 70:346-350.
Costarelli L, Muti E, Malavolta M, Giacconi R, Cipriano C, Sartini
D, Emanuelli M, Silvestrini M, Provinciali L, Gobbi B &
Mocchegiani E (2008). Modulation of genes involved in Zinc
homeostasis in old low-grade atherosclerotic patients under effects
of HMG-CoA reductase inhibitors. Rejuvenation Res 11:287-291.
Desai A, ZhaoY & Warren JS (2008). Development of atherosclerosis
in Balb/c apolipoprotein E-deficient mice. Cardiovasc Pathol
(Epub ahead of print).
Formigari A, Irato P & Santon A (2007). Zinc, antioxidant systems
an dmetallothionein in metal mediated-apoptosis: Biochemical and
cytochemical aspects. Com Biochem Physiol C Toxicol Pharmacol
(Epub ahead of print).
Hennig B, Wang Y, Ramasamy S & McClain CJ (1992). Zinc
deficiency alters barrier function of cultured porcine endothelial
cells. J Nutr 122:1242-1247.
Hennig B, Meerarani P, Ramadass P, Toborek M, Malecki N, Slim
R & McClain CJ (1999). Zinc nutrition and apoptosis of vascular
endothelial cells: implications in atherosclerosis. Nutrition
Henriksen PA & Sallenave JM (2008). Medicine in focus Human
neutrophil elastase: Mediator and therapeutic target in atheros-
clerosis. Int J Biochem Cell Biol 40:1095-1100.
Young-Eun Cho et al. Download full-text
Kharbanda R & MacAllister RJ (2005). The Atherosclerosis time-line
and the role of the endothelium. Endocr Metab Agents 5:47-52.
Kunsch C & Medford RM (1999). Oxidative stress as a regulator of
gene expression in the vasculature. Circ Res 85 (8):753-766.
Ling S, Nheu L , Dai A, Guo Z & Komesaroff P ( 2007). Effects
of four medicinal herbs on human vascular endothelial cells in
culture. Int J Cardiol (Epub ahead of print).
McClain C, Morris P & Hennig B (1995). Zinc and endothelial
function. Nutrition 11:117-120.
Minqin R, En H, K Beck, R Rajendran, Wu BJ, Halliwell B, Watt
F & Stocker R (2007). Nuclear microprobe investigation into the
trace elemental contents of carotid artery walls of apolipoprotein
E deficient mice. Nucl Instrum Methods Phys Res B 260:240-244.
Meerarani P, Ramadass P, Toborek M, Bauer HC, Bauer H & Hennig
B (2000). Zinc protect against apoptosis of endothelial cells
induced by linoleic acid and tumor necrosis factor alpha. Am J
Clin Nutr 71:81-87.
Papagianni A, Kalovoulos M, Kirmizis D, Vainas A, Belechri A,
Alexopoulos E & Memmos D (2003). Carotid atherosclerosis is
associated with inflammation and endothelial cell adhesion
molecules in chronic haemodialysis patients. Nephrol Dial
Reiterer G, Toborek M & Hennig B (2004). Peroxisome proliferator
activated receptors and γ require zinc for their anti-inflammatory
properties in porcine vascular endothelial cells. J Nutr 134:1711-
Ren M, Watt F, Huat BTK & Halliwell B (2003). Correlation of iron
and zinc levels with lesion depth in newly formed atherosclerotic
lesions. Free Radic Biol Med 34:746-752.
Theilmeier G, Michiels C, Spaepen E, Vreys I, Collen D, Vermylen
J & Hoylaerts MF. (2008). Endothelial von Willebrand factor
recruits platelets to atherosclerosis-prone sites in response to
hypercholesterolemia. Blood 99:4486-4493.
Watt F, Rajendran R, Ren MQ, Tan BKH & Halliwell B (2006). A
nuclear microscopy study of trace elements Ca, Fe, Zn and Cu
in atherosclerosis. Nucl Instrum Methods Phys Res 249:646-652.
Yang H, Roberts LJ, Shi MJ, Zhou LC, Ballard BR, Richardson A
& Guo ZM (2004). Retardation of atherosclerosis by overexp-
ression of catalase or both Cu/Zn-superoxide dismutase and
catalase in mice lacking apolipoprotein. E. Circ Res 95:1075-1081.
Zheng HT, Zhou LN, Huang CJ, Hua X, Jian R, Su BH & Fanga
F (2008). Selenium inhibits high glucose and high insulin-induced
adhesion molecule expression in vascular endothelial cells. Arch
Med Res 39:373-379.