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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 959-967
Homocysteine-Induced Endoplasmic Reticulum Stress and Growth Arrest
Leads to Specific Changes in Gene Expression in Human Vascular
Endothelial Cells
By
P. Andrew Outinen,
Sudesh K. Sood,
Sabine I. Pfeifer,
Sushmita Pamidi,
Thomas J. Podor,
Jun Li,
Jeffrey I. Weitz, and
Richard C. Austin
From the Department of Pathology and Molecular Medicine, McMaster
University and the Hamilton Civic Hospitals Research Centre, Hamilton,
Ontario, Canada.
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ABSTRACT |
Alterations in the cellular redox potential by homocysteine promote
endothelial cell (EC) dysfunction, an early event in the progression of
atherothrombotic disease. In this study, we demonstrate that
homocysteine causes endoplasmic reticulum (ER) stress and growth arrest
in human umbilical vein endothelial cells (HUVEC). To determine if
these effects reflect specific changes in gene expression, cDNA
microarrays were screened using radiolabeled cDNA probes generated from
mRNA derived from HUVEC, cultured in the absence or presence of
homocysteine. Good correlation was observed between expression profiles
determined by this method and by Northern blotting. Consistent with its
adverse effects on the ER, homocysteine alters the expression of genes
sensitive to ER stress (ie, GADD45, GADD153, ATF-4, YY1). Several other genes observed to be differentially expressed by homocysteine are known
to mediate cell growth and differentiation (ie, GADD45, GADD153, Id-1,
cyclin D1, FRA-2), a finding that supports the observation that
homocysteine causes a dose-dependent decrease in DNA synthesis in
HUVEC. Additional gene profiles also show that homocysteine decreases
cellular antioxidant potential (glutathione peroxidase, NKEF-B PAG,
superoxide dismutase, clusterin), which could potentially enhance the
cytotoxic effects of agents or conditions known to cause oxidative
damage. These results successfully demonstrate the use of cDNA
microarrays in identifying homocysteine-respondent genes and indicate
that homocysteine-induced ER stress and growth arrest reflect specific
changes in gene expression in human vascular EC.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HYPERHOMOCYST(E)INEMIA (HH) is a
significant independent risk factor for premature atherothrombotic
disease.1-5 Up to 40% of patients diagnosed with coronary,
cerebrovascular, or peripheral atherosclerosis have HH.3-6
Although the majority of cases of HH are thought to be caused by an
interplay between dietary and genetic factors, the genetic disorders
are associated with the highest plasma levels of homocysteine, with
cystathionine  -synthase deficiency (CBS)4-6 and
5,10-methylenetetrahydrofolate reductase deficiency7,8
being the most common. Regardless of the underlying cause of HH, the
relationship between elevated blood homocysteine levels and premature
vascular and thrombotic disease persists.
Earlier studies suggest that atherothrombosis associated with HH
reflects endothelial cell (EC) injury and/or dysfunction. Homocysteine
causes EC injury when administered to baboons9,10 or
rats,11 or when added directly to cultured
EC.12-15 Furthermore, deGroot et al14 showed
that primary cultures of EC obtained from obligate heterozygotes for
CBS deficiency are more sensitive to homocysteine-induced damage than
control cells. In addition to causing injury, homocysteine has been
shown to increase the procoagulant activity of cultured EC by (1)
inducing a protease that activates factor V,16 (2)
inhibiting protein C activation,17 (3) causing aberrant
processing of thrombomodulin (TM),18 (4) inducing tissue
factor activity,19 and (5) inhibiting the cellular binding
sites for tissue plasminogen activator.20 Homocysteine also
reduces nitric oxide production in vitro,21 a finding that could explain why diet-induced HH in monkeys22 and
pigs23 causes impaired vasomotor regulatory function.
Currently, the mechanism by which elevated levels of homocysteine cause
EC injury and/or dysfunction is relatively unknown. We, and others,
have demonstrated in cultured human vascular EC that homocysteine
increases the expression and synthesis of GRP78, an endoplasmic
reticulum (ER)-resident chaperon and member of the 70-Kd heat-shock
protein (HSP) family.24-26 In support of these in vitro
findings, steady-state mRNA levels of GRP78 were also shown to be
elevated in the livers of CBS-deficient mice that had HH.26
Given that GRP78 is induced by agents or conditions known to elicit ER
stress27 and that homocysteine impairs protein processing
and secretion via the ER,18,28 these studies suggest that
homocysteine alters the cellular redox state, thereby causing ER stress
and leading to growth arrest.29 Whether these effects of
homocysteine reflect specific changes in gene expression is not
completely known.
To explore this possibility, cDNA microarrays were screened using
radiolabeled cDNA probes generated from mRNA transcripts from human
vascular EC cultured in the absence or presence of homocysteine.
Herein, we provide a first step in identifying the gene pathways by
which homocysteine alters EC function and show that
homocysteine-induced ER stress and growth arrest reflect changes in
gene expression specific for these effects. Furthermore, additional
gene pathways demonstrate that homocysteine decreases the expression of
a wide range of antioxidant enzymes that could enhance the adverse
effects of agents and/or conditions known to cause oxidative damage.
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MATERIALS AND METHODS |
Cell culture and treatment conditions.
Primary human umbilical vein endothelial cells (HUVEC) were isolated by
collagenase treatment of human umbilical veins30 and
cultured in EC medium (M199 medium containing 20% fetal bovine serum,
20 µg/mL EC growth factor, 90 µg/mL porcine intestinal heparin, 100 µg/mL penicillin, and 100 µg/mL streptomycin) in a humidified
incubator at 37°C with 5% CO2. Cells from passages 2 to 4 were used in these studies. The transformed HUVEC line, ECV304,
was obtained from the American Type Culture Collection (ATCC;
Rockville, MD) and cultured in EC medium. DL-Homocysteine or
dithiothreitol (DTT) (Sigma, St Louis, MO) was prepared in EC medium,
sterilized by filtration, and added to the cell cultures.
[3H]thymidine incorporation.
HUVEC were grown to 70% confluency in EC medium in the absence or
presence of increasing concentrations of homocysteine or DTT (0.2 to
5.0 mmol/L) for 18 hours. During the last 4 hours of treatment, cells
were labeled with [methyl-3H]thymidine (NEN Life
Sciences, Guelph, Canada) at 1 µCi/mL. After labeling, cells were
washed 3 times with phosphate-buffered saline (PBS), fixed in ice-cold
10% acetic acid, and washed with 95% ethanol. Incorporated
[3H]thymidine was extracted in 0.2N NaOH and measured in
a scintillation counter. Values were expressed as the mean ± SD
from 3 separate experiments.
Metabolic labeling and immunoprecipitation.
HUVEC grown to 70% confluence in EC medium were cultured in the
absence or presence of increasing concentrations of homocysteine (0.2 to 5.0 mmol/L) for 8 or 18 hours. Cells were then metabolically labeled
with 300 µCi/flask of EXPRE35S35S labeling
mix (NEN) in Dulbecco's modified Eagle's medium (DMEM) without
methionine or cysteine for 1 hour. Cell lysates were prepared in 1 mL
of lysis buffer (1% Triton-X100 in 25 mmol/L Tris, 100 mmol/L NaCl,
0.02% bovine serum albumin, 10 mmol/L EDTA, pH 8.0) that contained
protease inhibitors, centrifuged to precipitate insoluble material, and
incubated with protein A-Sepharose beads (Amersham Pharmacia
Biotech, Mississauga, Canada) to remove any material that binds
nonspecifically to protein A. Samples were then incubated with either
goat anti-von Willebrand factor (anti-vWF) antibodies (Affinity
Biologicals, Hamilton, Canada) or control goat IgG in the presence of
protein A-Sepharose overnight at 4°C. After washing with PBS, the
beads were boiled in sodium dodecyl sulfate polyacrylamide gel
electropheresis (SDS-PAGE) sample buffer (50 mmol/L Tris, pH 6.8, 2%
SDS, 10% glycerol, 1% -mercaptoethanol, 0.01% bromophenol blue)
and proteins separated on 7.5% SDS-polyacrylamide gels under reducing
conditions. Radiolabeled proteins were detected by fluorography after
incubation with Amplify (Amersham Pharmacia Biotech).
vWF multimer analysis.
Whole-cell lysates were prepared by denaturation of HUVEC in lysis
buffer that contained 1 mg/µL urea and 0.1% SDS. vWF multimers were
separated on 1.2% agarose gels in 1X electrophoresis buffer (50 mmol/L
Tris, 0.384 mol/L glycine, 0.1% SDS, pH 8.3) run at 35 V
overnight or until the buffer front was within 1 to 2 cm from the
anodal end of the gel. For detection of vWF, proteins were
electrophoretically transferred to nitrocellulose membranes, immunoblotted with anti-vWF antibodies, and visualized using the Renaissance chemiluminescence reagent (NEN).
Immunohistochemistry and image analysis.
Immunohistochemistry and image analysis was performed as described
previously.26 Briefly, after fixation and permeabilization, HUVEC were immunostained with either anti-GRP78 (StressGen, Vancouver, Canada) or anti-vWF antibodies and images captured using Northern Exposure image analysis/archival software (Mississauga, Canada).
Preparation of total RNA.
Total RNA was isolated from cells using the RNeasy total RNA kit
(Qiagen, Santa Clarita, CA) and resuspended in diethyl pyrocarbonate (DEPC)-treated water. Quantification and purity of the RNA was assessed
by A260/A280 absorption, and RNA samples with
ratios greater than 1.6 were stored at  70°C for further analysis.
Analysis of differential gene expression using a human cDNA
microarray.
Poly (A)+ RNA was isolated from total RNA using Oligotex
resin (Qiagen), as described previously.26 To generate
radiolabeled cDNA probes, poly (A)+ RNA was
reverse-transcribed with Moloney murine leukemia virus (MMLV) in the
presence of [ -32P]dATP (NEN). The radiolabeled cDNA
probes were purified from unincorporated nucleotides by gel filtration
in Chroma Spin-200 columns (Clontech, Palo Alto, CA) and hybridized
overnight at 68°C to a human cDNA microarray consisting of 588 known human genes under tight transcriptional control, as described by
the manufacturer (Clontech). After a series of high-stringency washes (three 20-minute washes in 2X saline-sodium citrate
[SSC], 1% SDS followed by two 20-minute washes in 0.1X
SSC, 0.5% SDS) at 68°C, the membranes were exposed to x-ray film
(Reflections NEF-496, NEN) and subjected to autoradiography. Changes in
gene expression were quantified by densitometric scanning of the
membranes using the ImageMaster VDS and Analysis Software (Amersham
Pharmacia Biotech).
Northern blot analysis.
Total RNA (10 µg/lane) was fractionated on 2.2 mol/L
formaldehyde/1.2% agarose gels and transferred overnight onto
Zeta-Probe GT nylon membranes (Bio-Rad, Toronto, Canada) in 10X SSC.
The RNA was cross-linked to the membrane using a UV crosslinker (PDI Bioscience, Toronto, Canada) before hybridization. Specific probes were
generated by labeling the cDNA fragments with
[-32P]dCTP (NEN) using a random primed DNA labeling
kit (Boehringer Mannheim, Laval, Canada). After overnight hybridization
at 43°C, the membranes were washed as described by the
manufacturer, exposed to x-ray film, and subjected to autoradiography.
Changes in steady-state mRNA levels were quantified by densitometric
scanning of the blots as described earlier. To correct for differences
in gel loading, integrated optical densities were normalized to human
glyceraldehyde 3-phosphate dehydrogenase (GAPDH). cDNA probes encoding
Id-1 (GenBank/EBI accession no. AA582212), natural killer
cell-enhancing factor B (NKEF-B) (no. AA100194),
proliferation-associated protein (PAG) (no. AA204896), sterol
regulatory element-binding protein 1 (SREBP) (no. AA568572), clusterin
(no. AA614020), growth arrest and DNA damage-inducible (GADD) genes,
GADD153 (no. AA627477) and GADD45 (no. AA147214) were obtained from
Genome Systems (St Louis, MO). The cDNA probe encoding GRP78 has been
described previously.26
Statistical analysis.
Data are presented as the mean ± SD. Statistical differences
between the homocysteine-treated and control groups were determined by
analysis of variance. If a significant difference between treated and
control groups was demonstrated, an unpaired Student's t-test was performed for each point. For all analyses, P values less than .05 were considered statistically significant.
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RESULTS |
Effect of homocysteine on HUVEC viability and growth.
To examine the effect of homocysteine on cell viability,
51Cr release assays were performed as described
previously.14 Exposure of HUVEC to 5 mmol/L homocysteine
for up to 18 hours had no significant effect on overall cell viability,
compared with control cells (19.3% ± 0.5% v 17.4% ± 0.6% release, respectively, at 6 hours, n = 3; 27.9% ± 2.3%
v 28.1% ± 0.7% release, respectively, at 18 hours, n = 3). The time-dependent basal release of 51Cr from HUVEC is
consistent with previous studies.14 In contrast to 5 mmol/L
homocysteine alone, 5 mmol/L homocysteine in the presence of
4 µmol/L Cu2+, which is known to generate
H2O2 and induce EC lysis,12-15
caused a significant increase (P > .01) in the release of
51Cr at 18 hours (28.1% ± 0.7% v 81.4% ± 1.2% release, respectively, n = 3).
To determine the effect of homocysteine on cell proliferation, HUVEC
were cultured in the absence or presence of various concentrations of
homocysteine and [3H]thymidine incorporation was
measured. As shown in Fig 1, HUVEC exposed
to homocysteine for 18 hours demonstrated a dose-dependent decrease in
DNA synthesis and is consistent with earlier studies.29 In
addition to homocysteine, the thiol-containing reducing agent DTT also
caused a similar dose-dependent decrease in DNA synthesis (data not
shown).
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| Fig 1.
Effect of homocysteine on DNA synthesis. Primary HUVEC
were incubated in EC medium in the absence or presence of increasing
concentrations of homocysteine for 18 hours.
[3H]thymidine incorporation was measured during the last
4 hours. Values represent the mean ± SD from 3 wells from 3 separate
experiments. * P < .05 v untreated HUVEC, **P
< .01.
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Homocysteine induces ER stress in HUVEC.
Previous studies have shown that intracellular transport of vWF and TM
via the ER is selectively inhibited by homocysteine in
HUVEC.18,28 Homocysteine also increases the expression and synthesis of GRP78, a resident ER chaperon induced by agents or conditions known to adversely affect ER function.24-26 To
further investigate the effect of homocysteine on ER function, vWF
processing and secretion, and its interaction with GRP78 were examined
in HUVEC cultured in the absence or presence of homocysteine.
The effect of homocysteine on intracellular levels and distribution of
GRP78 and vWF in HUVEC was examined by indirect immunofluorescence using anti-vWF or anti-GRP78 antibodies. In control HUVEC, GRP78 was
concentrated in the perinuclear region, consistent with its presence in
the ER (Fig 2A). In contrast, both the
distribution and intensity of GRP78 immunostaining was markedly
enhanced in HUVEC exposed to homocysteine (Fig 2B). Unlike GRP78, vWF
immunostaining and localization were dramatically reduced in HUVEC
exposed to homocysteine (Fig 2E), compared with control cells (Fig 2D).
As controls, no specific staining was observed in untreated HUVEC incubated with either normal mouse (Fig 2C) or rabbit IgG (Fig 2F).
Consistent with these findings, intracellular levels of vWF dimers were
dramatically decreased after 4 hours in HUVEC exposed to 5 mmol/L
homocysteine (Fig 3).
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| Fig 2.
Effect of homocysteine on the cellular localization of
GRP78 and vWF. HUVEC plated on gelatin-coated glass coverslips were
cultured in the absence (A,D) or presence (B,E) of 5.0 mmol/L
homocysteine for 18 hours. After treatment, cells were fixed,
permeabilized, and incubated with antibodies against either GRP78 (A,B)
or vWF (D,E). Antibodies were subsequently detected using
fluorescein-labeled secondary antibodies. Parallel experiments using
normal mouse (C) or rabbit IgG (D) were performed to assess nonspecific
immunofluorescence.
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| Fig 3.
Effect of homocysteine on vWF multimerization. HUVEC
cultured in the absence or presence of 5.0 mmol/L homocysteine for 1, 2, 4, 8, or 18 hours were denatured in lysis buffer, the lysates
fractionated on 1.2% (wt/vol) agarose gels and transferred to
nitrocellulose. vWF monomers, dimers, and multimers were detected by
immunoblotting using anti-vWF antibodies.
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Stable association of GRP78 with misfolded, improperly glycosylated, or
incompletely assembled proteins in the ER leads to retention and
intracellular degradation.31-33 To determine whether GRP78
was capable of stably binding to misfolded vWF within the ER, HUVEC
exposed to various concentrations of homocysteine for 8 or 18 hours
were metabolically labeled with [35S]cysteine and
[35S]methionine, and vWF from cell lysates was
immunoprecipitated with anti-vWF antibodies as described in the
Methods. Bands that corresponded to mature and pro-vWF were detected in
immunoprecipitated eluates from HUVEC treated without or with
homocysteine (Fig 4). However, coimmunoprecipitation of
vWF with GRP78 was observed only in HUVEC treated with 1 or 5 mmol/L
homocysteine for 8 hours or 5 mmol/L homocysteine for 18 hours. Taken
together, these findings imply that GRP78 binds to misfolded vWF,
prevents its secretion from the ER and likely directs aberrantly folded
vWF to the degradative machinery.
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| Fig 4.
Homocysteine increases the stable association between
GRP78 and vWF. HUVEC cultured in the absence or presence of 0.2, 1.0, or 5.0 mmol/L homocysteine for 8 or 18 hours were metabolically labeled
for 1 hour in the presence of [35S]methionine and
[35S]cysteine. Radiolabeled proteins from cell lysates
were immunoprecipitated with anti-vWF antibodies, separated on 7.5%
SDS polyacrylamide gel under reducing conditions, and detected by
fluorography. Immunoprecipitation of pro and mature vWF coprecipitated
GRP78. kD, molecular mass markers.
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Effect of homocysteine on differential gene expression in HUVEC.
Previous studies have shown that cDNA arrays provide a rapid and
effective method in monitoring differential gene
expression.34-38 To investigate the possibility that
homocysteine-induced ER stress and growth arrest reflect
specific changes in gene expression in ECs, a human cDNA microarray
that contained 588 known human genes was screened using radiolabeled
cDNA generated from total poly (A)+ RNA from primary HUVEC
cultured in the absence or presence of 5 mmol/L homocysteine for 4 or
18 hours. After hybridization, the cDNA array membranes were washed
under high stringency and the hybridization patterns analyzed by
autoradiography. The level of nonspecific hybridization was low since
the negative DNA controls, including M13mp18(+) strand DNA,  DNA, and
pUC18, failed to show any hybridization signal. To ensure accurate
comparisons in the expression levels of each gene on the cDNA array,
hybridization signals were normalized to the signals obtained from
housekeeping gene controls (ie, ubiquitin, glyceraldehyde 3-phosphate
dehydrogenase, -tubulin, human leukocyte antigen [HLA] class 1 histocompatibility antigen C-4, -actin, 23-Kd highly basic protein,
ribosomal protein S9) on the same array.
As shown in Fig 5, the hybridization
patterns between wild-type and homocysteine-treated HUVEC were similar.
However, analysis of the cDNA microarray showed that a total of 16 of
588 (2.7%) of the known human genes were differentially expressed in
primary HUVEC exposed to 5 mmol/L homocysteine for 4 or 18 hours (Table 1). Similar changes in gene expression were
also observed in the ECV304 cell line cultured in the absence or
presence of homocysteine (data not shown). The percentage change in
gene expression observed in this study is consistent with the recent
observation that 2.5% of genes are regulated in HepG2 hepatoma cells
exposed to -mercaptoethanol, another thiol-containing agent known to
cause ER stress and growth arrest.39 Of these genes, 9 were
shown to be induced by homocysteine, while 7 were downregulated. Among
the genes detected with significantly higher expression (>10-fold)
was GADD153, a stress-response gene known to be induced by agents or
conditions that adversely affect ER function.40 ATF-4, a
stress-inducible transcription factor, was also shown to be induced by
homocysteine and is consistent with earlier studies using mRNA
differential display to identify ATF-4 as a homocysteine-inducible
gene.24 Other inducible genes included Id-1, guanine
nucleotide-binding protein G-S, SREBP, transcriptional repressor
protein YY1, and the transcription factor ETR103. Genes shown to be
downregulated by homocysteine included the antioxidant enzymes NKEF-B,
superoxide dismutase, glutathione peroxidase, clusterin, and PAG, as
well as FRA-2, adenosine triphosphate (ATP)-dependent DNA helicase, and
cyclin D1. The observation that the majority of significant changes in
gene expression occurred at 4 hours and declined by 18 hours suggests
that homocysteine causes an initial early response in EC gene
expression, followed by an adaptive response that likely involves
cellular factors that influence the metabolism or elimination of
intracellular homocysteine.
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| Fig 5.
cDNA microarray analysis of changes in gene expression in
primary HUVEC cultured in the absence or presence of homocysteine.
[32P]-labeled cDNA probes generated from poly
(A)+ RNA from control HUVEC (A) or HUVEC exposed to
5 mmol/L homocysteine for either 4 (B) or 18 hours (C) were hybridized
to a cDNA microarray containing 588 known human genes. After a series
of high-stringency washes, hybridization patterns were analyzed by
autoradiography. Upward or downward arrows indicate the location
within the array of genes increased or decreased, respectively, by
homocysteine at 4 or 18 hours, v untreated cells. The relative
expression levels of specific cDNAs was assessed by comparison with a
wide range of housekeeping genes. Results are representative of 2 separate hybridization experiments.
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Table 1.
Identification of Homocysteine-Respondent Genes Using
cDNA Microarrays in Primary HUVECs Exposed to 5 mmol/L Homocysteine for
4 or 18 Hours
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To test the reliability of the cDNA microarrays in identifying
differentially expressed genes, we analyzed 7 different
homocysteine-responsive genes by Northern blot analysis (Fig
6A). In each case, the relative expression
levels of these homocysteine-responsive genes observed on Northern
blots was consistent with the differential gene expression identified
by microarray hybridization (Table 2). As a
positive control, homocysteine was shown to induce the expression of
GRP78 (Fig 6B), a finding consistent with our earlier
studies.26
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| Fig 6.
Northern blot analysis substantiating the
consistency of the cDNA microarray results. Total RNA (10 µg/lane)
isolated from HUVEC cultured in the absence or presence of homocysteine
for the indicated time periods was analyzed by Northern blot
hybridization, followed by autoradiography. (A) Effect of homocysteine
on Id-1, NKEFB, clusterin, PAG, SREBP, GADD153, and GADD45 mRNA levels.
(B) Effect of homocysteine on GRP78 mRNA levels.
Hybridization to a human GAPDH probe was used to normalize RNA loading.
The results are representative of 2 separate experiments using 2 different samples of RNA.
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DISCUSSION |
Previous studies using cultured human vascular EC have shown that
alterations in the cellular redox potential by homocysteine impair
protein processing and secretion via the ER18,28 and increase expression of the ER stress-response gene,
GRP78.24-26 In this study, we demonstrate that homocysteine
causes ER stress in cultured human vascular EC by (1) decreasing
intracellular levels of vWF, (2) inducing the expression and synthesis
of GRP78, and (3) increasing the stable association of vWF with GRP78.
Furthermore, homocysteine was shown to cause a dose-dependent decrease
in DNA synthesis, a result consistent with earlier
findings.29 Taken together, these findings both support and
extend previous studies using cultured vascular
EC18,24-26,28 and suggest that homocysteine acts
intracellularly by altering the cellular redox state, thereby leading
to ER stress and growth arrest in EC.
To determine if homocysteine-induced ER stress and growth arrest
results in specific changes in gene expression in EC, cDNA microarrays
were screened. This approach was taken based on previous studies
showing that cDNA microarrays provide a powerful approach for studying
differential gene expression associated with the pathogenesis of cancer
and other diseases.34-38 As a result of this analysis, we
demonstrate that the effects of homocysteine on ER function and cell
growth reflect specific changes in gene expression. Furthermore,
additional gene profiles indicate that homocysteine suppresses the
ability of EC to protect themselves from agents or conditions known to
elicit oxidative damage.
Although relatively high concentrations of homocysteine ( 1 mmol/L)
were used to evaluate changes in gene expression, there was no effect
on overall cell viability, a finding consistent with previous studies
that indicated EC from normal individuals are relatively resistant to
high doses of homocysteine.12-19 This may reflect the fact
that only a small percentage of exogenous homocysteine (<1%) added
to the culture medium is actually taken up
intracellularly.41 Indeed, we have shown that intracellular concentrations of homocysteine are increased only 2-fold and 5-fold in
HUVEC exposed to 1 or 5 mmol/L homocysteine, respectively, compared
with untreated cells.26 Thus, the high doses required to
alter gene expression in vitro likely reflects the need to increase
intracellular levels of homocysteine by overcoming the cellular factors
that influence the metabolism and/or elimination of homocysteine.
The observation that homocysteine induces the expression of GADD45,
GADD153, ATF-4, and YY1 provides genetic evidence that homocysteine
causes ER stress. Although GADD45, a downstream effector of p53, and
GADD153, a member of the C/EBP gene family of transcription factors,42 have also been shown to be induced by growth
arrest, by DNA damage, or by UV irradiation,43-45 recent
studies indicate that inducers of GRP78 increase the expression of
these genes,46 and that the induction of GADD153 is more
responsive to ER stress.40 Based on previous studies
demonstrating that DTT, a thiol-containing agent known to cause ER
stress, induces the expression of both GRP78 and GADD153,47
our findings are not unexpected. Although the physiologic significance
of GADD gene induction by homocysteine has not been defined,
overexpression of the GADDs causes growth arrest in several cell
types.48,49 Furthermore, the importance of GADD153 in
cellular growth and differentiation comes from the molecular analysis
of human sarcomas wherein the rearrangement of the GADD153 gene gives
rise to a naturally occurring altered form of GADD153 incapable of
eliciting growth arrest.50 Given our findings, as in other
studies,29 that homocysteine inhibits EC growth, the GADDs
may play a potential role in linking ER stress to alterations in cell
growth and proliferation. This concept is also supported by our
observation that DTT, a known inducer of ER stress,27 also
decreases EC proliferation. The homocysteine-induced expression of
ATF-4, a member of the activating transcription factor/cyclic adenosine
monophosphate (cAMP)-responsive element-binding protein (ATF/CREB)
family of transcription factors, is consistent with previous
studies.24 ATF-4 is induced by increased intracellular Ca2+ concentrations51 and by
anoxia,52 conditions known to alter ER function. YY1, a
member of the GLI zinc finger family, has been shown to specifically
enhance the transcriptional activation of the GRP78 promoter under a
variety of ER stress conditions.53 These include depletion
of ER Ca2+ stores, inhibition of glycosylation and
formation of misfolded proteins. Thus, the ability homocysteine to
increase the expression of YY1 would not only enhance the expression of
GRP78, but could potentially mediate stress signals generated from the
ER to the nucleus. Given that YY1 affects cell growth54 and
acts as a transcriptional repressor,55 the induction of
YY1, like the GADD genes, by homocysteine may also play a role in
mediating EC growth.
Alterations in the expression of genes known to mediate cell growth and
differentiation is consistent with the finding that homocysteine causes
a dose-dependent decrease in EC growth. Overexpression of Id-1, a
member of the helix-loop-helix transcriptional
regulators,56 suppresses cell differentiation in mammary
epithelial cells57 and murine erythroleukemia
cells.58 The ability of homocysteine to decrease cyclin D1,
a positive growth regulator during the early G1
phase,27 and FRA-2, a transcription factor known to promote
osteoblast differentiation,59 also suggests that
homocysteine affects the expression of growth response genes in ECs.
Whether a decrease in cell growth plays a role in protecting EC from
homocysteine-induced injury and/or dysfunction is currently unknown.
The ability of homocysteine to inhibit the expression of the
antioxidant enzymes glutathione peroxidase and NKEF-B are consistent with our previous findings26 and suggest that homocysteine
may promote EC dysfunction and/or injury by indirectly enhancing the cytotoxic effect of agents or conditions that cause oxidative stress.
This concept is further supported by the recent observation that
homocysteine impairs the ability of glutathione peroxidase to detoxify
peroxides60 and acts synergistically with
H2O2 to enhance mitochondrial
damage.61 In addition to these antioxidant enzymes,
homocysteine decreased the expression of clusterin and PAG. Clusterin,
a multifunctional heterodimeric glycoprotein, has been implicated in a
wide range of physiologic functions such as lipid transport, tissue
repair and remodelling, membrane protection, and promotion of cell
interactions.62 Recent studies have also demonstrated that
induction of clusterin during the development of atherosclerosis may
represent a protective response to the oxidative stress associated with
the development of atherosclerosis.63,64 Furthermore,
because clusterin is a novel potent inhibitor of complementation-mediated cytolysis, the ability of homocysteine to
decrease clusterin gene expression could potentially enhance complement
activation at the site of vessel wall damage, which results in
increased EC injury and/or dysfunction. PAG, a novel antioxidative
protein family member responsive to oxidative stress,65-67 induces cell growth and differentiation by blocking c-Abl
tyrosine kinase activity. The ability of homocysteine to decrease
PAG expression would not only enhance the cytotoxic effects of oxidants
but could suppress EC growth and differentiation.
Although we have identified several gene pathways by which homocysteine
could potentially influence EC function and growth, a number of
relevant issues remain to be explored. Additional studies are needed to
determine if these homocysteine-dependent changes in gene expression in
cultured vascular EC are observed in vivo. The fact that GRP78 is
induced in the livers of CBS-deficient mice,26 and that the
activity of several antioxidant enzymes is altered in rabbits fed a
high methionine diet68 supports our findings and suggests
that our in vitro studies likely reflect the actions of homocysteine in
vivo. Furthermore, it is not known if the ability of homocysteine to
cause ER stress directly influences EC growth. Based on the observation
that agents known to adversely affect ER function cause inhibition of
protein synthesis69 and cell growth,70-72 it is
likely that elevated levels of intracellular homocysteine act in a
similar fashion. As a result of previous studies demonstrating that
homocysteine induces smooth muscle cell proliferation and increases the
expression of the cyclin genes,29 it will be of interest to
determine if the changes in gene expression observed in smooth muscle
cells by homocysteine inversely correlate with some of the genes
identified in these studies.
In summary, we have shown that homocysteine-induced ER stress and
growth arrest in vascular EC involves changes in gene expression specific for these effects. Furthermore, the ability of homocysteine to
decrease the expression of several antioxidant enzymes suggests that
homocysteine could indirectly enhance the effects of agents or
conditions known to cause oxidative stress. We also show the utility of
cDNA microarrays as an initial screening approach for the
identification of homocysteine-respondent genes in EC. Based on the
high yield of information obtained using an array of fewer than 600 known human genes, a more comprehensive survey of gene expression
patterns, using a more complete array of human genes, will not only
provide additional important information on the mechanism by which
homocysteine promotes EC dysfunction but will also increase our
understanding of the gene pathways involved in the pathogenesis of HH.
| |
ACKNOWLEDGMENT |
We thank Dr Catherine Hayward for technical assistance in analyzing the
vWF multimers.
| |
FOOTNOTES |
Submitted August 24, 1998; accepted March 29, 1999.
Supported by Research Grant No. NA-3307 from the Heart and Stroke
Foundation of Ontario (to R.C.A.). P.A.O. was a recipient of a Douglas
C. Russel Memorial Scholarship and an Ontario Graduate Scholarship.
R.C.A. is a Research Scholar of the R.K. Fraser Foundation.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Richard C. Austin, PhD, Hamilton Civic
Hospitals Research Centre, 711 Concession St, Hamilton, Ontario,
Canada, L8V 1C3; e-mail: raustin{at}thrombosis.hhscr.org.
| |
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TOP
ABSTRACT
INTRODUCTION