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Weill Medical College of Cornell University, Departments of Pediatrics and Medicine, New York, NY 10021
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT Homocysteine impairs endothelial...Homocysteine inhibits...Homocysteine induces expression...REFERENCES |
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, ß, 
and 
by homocysteine is associated with increased
turnover of at least one free thiol–containing protein, suggesting
that up-regulation of these subunits may represent a mechanism for
replacement of damaged or modified proteins. A more complete
understanding of the diverse effects of homocysteine on endothelial
cell function may provide important clues to the precise role
homocysteine may play in the initiation and progression of vascular
disease.
KEY WORDS: • homocysteine elongation factors • endothelial cell • tissue plasminogen activator • plasmin
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Homocysteine impairs endothelial cell thromboresistance |
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TOPABSTRACTHomocysteine impairs endothelial...Homocysteine inhibits...Homocysteine induces expression...REFERENCES |
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). For example, there is an association between exogenously applied HC
and inhibition of prostacyclin synthesis (Wang et al. 1993
), activation of procoagulant factor V (Rodgers and Kane 1986
), impaired activation of the natural anticoagulant
protein C (Rodgers and Conn 1990
) and
down-regulation of cell surface thrombomodulin (Hayashi et al. 1992
, Lentz and Sadler 1991
). Additional
studies have shown impaired regulation of nitric oxide (Stamler et al. 1993
), induction of tissue factor (Fryer et al. 1993
) and suppression of heparan sulfate expression
(Nishinaga et al. 1993
). Many of these studies, however,
employed supraphysiologic levels of HC; thus, they must be interpreted
with caution. In one salient in vivo study, monkeys made mildly
hyperhomocyst(e)inemic (10 µmol/L) by dietary manipulation
showed abnormal vasoconstriction and reduced thrombomodulin activity in
the aorta (Lentz et al. 1996
). The mechanisms by which
HC may exert these diverse effects are largely unclear, as is the
question of whether functional modifications observed in vitro also
apply in vivo.
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Homocysteine inhibits endothelial cell surface fibrinolytic assembly |
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TOPABSTRACTHomocysteine impairs endothelial...Homocysteine inhibits...Homocysteine induces expression...REFERENCES |
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). This system consists of a cascade of proteolytic
reactions oriented largely on fibrin-containing surfaces, i.e.,
thrombi, or on the surfaces of cells. Plasminogen is a 93-kDa
glycoprotein that circulates in plasma at a concentration of ~1.5
µmol/L. Itself inactive, plasminogen is converted to the
functional serine protease plasmin upon cleavage of a single peptide
bond at position 560–561 (Arg-Val). This hydrolysis can be carried out
by either of two major plasminogen activators, tissue plasminogen
activator (t-PA), a product of some endothelial cells, or urokinase
(u-PA), a product of renal epithelial cells. Fibrin, the principal
substrate of plasmin, is also the most potent known cofactor for
t-PA–dependent activation of plasminogen, accelerating its catalytic
efficiency ~500-fold. Further regulation of the system is provided by
an array of circulating inhibitors such as plasminogen activator
inhibitor-1 or 2-antiplasmin, which
immediately neutralize freely circulating t-PA and u-PA or
plasmin, respectively. Finally, fibrinolytic activity is localized to
cell surfaces through the interaction of plasminogen and its activators
with specific cell surface receptors.
Annexin II is a widely distributed, highly conserved, 36-kDa peripheral
membrane protein expressed abundantly on endothelial cells,
macrophages, myeloid cells and some tumor cells (Hajjar 1998
). All 20 known annexins consist of a conserved "core"
domain (30–40 kDa), which imparts phospholipid-binding capacity,
and a variable amino terminal "tail" domain (3–6 kDa) through
which specialized functions are acquired (Raynal and Pollard 1994
, Swairjo and Seaton 1994
). The human
annexin II gene consists of 13 exons distributed over 40 kb of genomic
DNA on chromosome 15 (15q21) (Spano et al. 1990
).
Annexin II possesses the unique property of binding both plasminogen
(Kd = 114 nmol/L) and tPA
(Kd = 30 nmol/L) (Hajjar and Hamel 1990
, Hajjar 1991
, Hajjar et al. 1994
), thereby stimulating the catalytic efficiency of
t-PA–dependent plasminogen activation 60-fold (Cesarman et al. 1994
) (Fig. 1
). This "fibrin-like" cofactor activity is inhibited completely in
the presence of lysine analogs or upon removal of its basic
carboxyl-terminal amino acids. Although it lacks a classical signal
peptide, annexin II is translocated constitutively to the endothelial
cell surface within 16 h of its biosynthesis. It binds to cell
surface phospholipid via core repeat 2 containing the linear sequence
KGLGT and downstream aspartate residue (Asp161) (Hajjar et al. 1996
). Annexin II heterotetramer, composed of two annexin
monomers and two p11 subunits, may have even greater stimulatory
effects on t-PA–dependent plasmin generation (Kassam et al. 1998
).
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).
Treatment of cultured endothelial cells with
DL-homocysteine is associated specifically with a 65%
reduction in functional binding sites for t-PA, whereas no effect
on plasminogen binding is observed (Hajjar 1993
). This
finding was associated with a 60% reduction in cell-associated
t-PA activity even though the intrinsic catalytic capability of
t-PA is not altered by HC. Binding of t-PA, but not
plasminogen, to annexin II monomer is also markedly reduced by ligand
blot analysis. These studies suggest that HC might perturb the
intrinsic fibrinolytic potential of the endothelial cell by selectively
blocking the t-PA binding domain of annexin II (Fig. 1)
.
tPA binding to annexin II depends upon a specific domain containing or
closely associated with residues 8–13 (LCKLSL) within the receptor’s
amino terminal "tail" domain (Hajjar et al. 1998
).
Recombinant annexin II, from which the tail domain has been cleaved,
fails to compete with immobilized intact annexin II for t-PA
binding. In addition, two overlapping dodecapeptides that mimic the
extreme amino terminal portions of the tail domain of annexin II
compete specifically and effectively with the intact protein for
t-PA binding. Similarly, the hexapeptide LCKLSL, but not the
mutated peptide LGKLSL, representing the region of overlap, blocks
binding of t-PA to both intact recombinant annexin II and
endothelial cell monolayers. These studies serve to pinpoint the tPA
binding domain of annexin II to residues 8–13 (LCKLSL) of the tail
domain.
The effect of HC on the t-PA-binding annexin II tail domain has been
examined in detail (Hajjar et al. 1998
). Electrospray
ionization mass spectrometric analysis of recombinant annexin II
indicated that HC alters the receptor physically, increasing its mass
by 135 ± 4 Da. Tandem mass spectrometric analysis of a tryptic
digest of HC-treated annexin II revealed derivatization of a single
cysteine residue (C9) within the amino terminal t-PA binding
domain. A disulfide-mediated complex between HC and annexin II was
also demonstrated in cultured endothelial cells that were metabolically
labeled with [35S]-HC. Modification of annexin
II by micromolar concentrations of HC was associated with a dose- and
time-related reduction in t-PA binding. Interestingly, the
I50 for inhibition of t-PA binding to annexin
II was ~11 µmol/L, a value close to the upper limit of
normal for HC in plasma (~14 µmol/L). Thus, inhibition
of t-PA–annexin II assembly on the endothelial cell, and subsequent
reduction in plasmin generation, could underlie the
prothrombotic/proatherogenic activity associated with HC in
vivo.
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Homocysteine induces expression of redox-adaptive genes |
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TOPABSTRACTHomocysteine impairs endothelial...Homocysteine inhibits...Homocysteine induces expression...REFERENCES |
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). We found a 3-7-fold,
time-dependent induction of a 220-bp fragment that demonstrated
complete sequence identity with elongation factor-1 (EF-1), a
member of a multimeric complex regulating mRNA translation.
HC-treated endothelial cells exhibited significantly increased
steady-state mRNA levels for EF-1, ß and , as well as
increased protein levels for EF-1, ß, and . These results
parallel recent studies in which tunicamycin and reducing
agent-responsive protein, the stress protein GRP78/BiP, activating
transcription factor 4 and a methylenetetrahydrofolate
dehydrogenase/cyclohydrolase were all induced adaptively in endothelial
cells exposed to high dose HC (Kokame et al. 1996
). In
vascular smooth muscle cells, the cyclin A gene also appears to be
activated transcriptionally after exposure to HC (Tsai et al. 1994
and 1996
).
In most eukaryotes, the two primary elongation factors, multimeric EF-1
and monomeric EF-2, are primary sites of regulation of protein
translation (Proud 1994
), and EF-1 appears to play a
crucial role in cell growth, proliferation and differentiation
(Merrick 1992
, Morris 1995
, Proud 1994
). EF-1 is a complex of polypeptides that regulate the
efficiency and fidelity of mRNA translation in eukaryotic cells. EF-1
consists of five subunits (2, ß, and
) that promote GTP-driven delivery of aminoacyl tRNAs to
the ribosome. The EF-1·GDP complex is converted to active
EF-1·GTP by the nucleotide exchange activities of EF-1ß and
EF-1. The EF-1 moiety is known to enhance the nucleotide exchange
activity of EF-1ß and may also serve to anchor the complex to
membrane structures. Interestingly, EF-1, which is homologous to
EF-1ß in the C-terminal nucleotide exchange region, is unique
among these factors in that it contains a leucine zipper motif of
unknown function.
Importantly, fibroblasts from cystathionine ß-synthase -/-
individuals also show a 1.5- to 3.0-fold increase in levels of mRNA for
EF-1, ß and , compared with normal cells (Chacko et al. 1998
). Treatment of normal cells with the HC precursor,
methionine, induced a 1.5- to 2.0-fold increase in EF-1, ß and
mRNA. These experiments demonstrate that EF-1 subunits are also
stimulated under conditions in which HC is generated intracellularly.
As judged by nuclear run-on studies, up-regulation of EF-1
subunits by HC reflects enhanced transcriptional activity in settings
in which HC is elevated. This induction, furthermore, was completely
inhibited by cycloheximide, suggesting a pathway by which HC may
stimulate synthesis of an intermediate protein, which then induces gene
transcription.
Finally, additional experiments suggest that free thiol–containing
proteins, such as annexin II, which may be susceptible to
derivatization upon exposure to HC (Hajjar et al. 1998
),
may experience increased rates of turnover compared with
"cysteineless" proteins (Chacko et al. 1998
).
Pulse-chase studies in HC-treated endothelial cells revealed a
doubling in the rate of appearance and disappearance of annexin II, but
no change in synthesis or degradation of plasminogen activator
inhibitor-1. These changes may represent an adaptive response to
HC-induced oxidative stress and protein degradation.
The precise role played by EF-1, ß, and in cells with
elevated levels of HC is unclear. On the one hand, HC may elicit an
increase in synthesis of a select population of polypeptides with which
it forms mixed disulfides. This hypothesis might require the presence
of a protein thiol-sensing mechanism that can recruit newly
synthesized translational cofactors and stimulate polypeptide
elongation. Alternatively, increased levels of EF-1 subunits might also
play a role in protein degradation because EF-1 has recently been
reported to have isopeptidase activity to promote the degradation of
N-acetylated proteins via the ubiquitin pathway
(Gonen et al. 1994
). Further studies in this area are
clearly required to resolve these issues.
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FOOTNOTES |
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3 Abbreviations used: EF, elongation factor; HC, homocysteine; t-PA, tissue plasminogen activator; u-PA, urokinase plasminogen activator.
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REFERENCES |
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TOPABSTRACTHomocysteine impairs endothelial...Homocysteine inhibits...Homocysteine induces expression...REFERENCES |
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