Clinical Biochemistry 36 (2003) 431– 441

Hyperhomocysteinemia and its role in the development of

atherosclerosis
A.B. Lawrence de Koning, Geoff H. Werstuck, Ji Zhou, Richard C. Austin*
Department of Pathology and Molecular Medicine, McMaster University and the Henderson Research Centre, Hamilton, Ontario, Canada

Received 31 December 2002; received in revised form 25 April 2003; accepted 25 April 2003

Abstract
Numerous epidemiological studies have demonstrated that hyperhomocysteinemia (HHcy) is a strong and independent risk factor for
cardiovascular disease. HHcy can result from a deficiency in the enzymes or vitamin cofactors required for homocysteine metabolism.
Several hypotheses have been proposed to explain the cellular mechanisms by which HHcy promotes cardiovascular disease, including
oxidative stress, endoplasmic reticulum (ER) stress and the activation of pro-inflammatory factors. Studies using genetic- and diet-induced
animal models of HHcy have now demonstrated a direct causal relationship between HHcy, endothelial dysfunction and accelerated
atherosclerosis. These recently established animal models of HHcy provide investigators with important in vivo tools to (i) further
understand the cellular mechanisms by which HHcy contributes to endothelial dysfunction and atherosclerosis, and (ii) develop therapeutic
agents useful in the treatment of cardiovascular disease. © 2003 The Canadian Society of Clinical Chemists. All rights reserved.

Keywords: Hyperhomocysteinemia; Atherosclerosis; Endothelial dysfunction; Endoplasmic reticulum stress

1. Introduction ischemic symptoms, acute coronary syndromes usually re-

Atherosclerosis, the principal cause of cardiovascular
disease and stroke, is a complex, chronic process that is
initiated at sites of endothelial cell injury and culminates in
the formation of stratified lesions of the arterial wall [1–5].
Subendothelial infiltration of monocytic cells, proliferation
and migration of smooth muscle cells, cholesterol deposi-
tion, and elaboration of extracellular matrix are hallmark
features of atherosclerotic lesions. Cholesterol-laden
smooth muscle cells and macrophages, morphologically
recognized as foam cells, are observed at all stages of lesion
development [6,7]. The importance of cholesterol and its
oxidized derivatives in the pathogenesis of atherosclerosis is
supported by studies demonstrating the presence of choles-
terol within the lesions from humans and animals [8 –12].
Traditionally, cholesterol and its oxidized derivatives are
thought to accumulate in atherosclerotic lesions, thereby
contributing to the formation of advanced, multilayered
atheromas. Although advanced atherosclerotic lesions cause
progressive narrowing of the vessel lumen that can lead to
* Corresponding author. Tel.: ⫹1-905-527-2299 ext. 42628; fax: ⫹1-
905-575-2646.
E-mail address: raustin@thrombosis.hhscr.org (R.C. Austin).
sult from lesion rupture and thrombosis [1– 4].
Conventional risk factors for cardiovascular disease, includ-
ing hypercholesterolemia, hypertension, smoking and diabetes,
account for approximately 50% of all cases [1,2,12]. Evidence
now indicates that HHcy, which occurs in approximately 5 to
7% of the general population, is an important, independent risk
factor for atherosclerosis and thrombotic disease [13–20]. Fur-
thermore, up to 40% of patients diagnosed with premature
coronary artery disease, peripheral vascular disease or recur-
rent venous thrombosis have HHcy [13–18].
In this review article, we will summarize the genetic and
nutritional factors that induce HHcy and further examine the
clinical evidence implicating HHcy as an independent risk
factor for cardiovascular disease. In addition, potential
mechanisms by which homocysteine accelerates atheroscle-
rosis will be discussed in light of the important findings
recently reported for both genetic- and diet-induced animal
models of HHcy.
2. Genetic and nutritional factors causing HHcy
Homocysteine is a thiol-containing amino acid that is
formed during the metabolic conversion of methionine to

0009-9120/03/$ – see front matter © 2003 The Canadian Society of Clinical Chemists. All rights reserved.
doi:10.1016/S0009-9120(03)00062-6
432 A.B. Lawrence de Koning et al. / Clinical Biochemistry 36 (2003) 431– 441
Fig. 1. Metabolism of homocysteine. Dietary methionine is converted to
the methyl donor S-adenosylmethionine (SAM) and is demethylated to
S-adenosylhomocysteine (SAH), which is subsequently cleaved into aden-
osine and homocysteine. Through the transsulphuration pathway, homo-
cysteine is converted to cystathionine and cysteine by the enzyme cysta-
thionine ␤-synthase (CBS) and the cofactor vitamin B6 (methylcobalamin).
Homocysteine can also be remethylated through the folate cycle. This
pathway depends on the enzyme methionine synthase (MS) and vitamin
B12 as well as the enzyme 5,10-methylenetetrahydrofolate reductase
(MTHFR) and folate, which enters the cycle as tetrahydrofolate (THF). In
liver and kidney, homocysteine is also remethylated by the enzyme betaine
homocysteine methyltransferase (BHMT), which transfers a methyl group
to homocysteine via demethylation of betaine to dimethylglycine (DMG).
cysteine (Figure 1). Once synthesized, homocysteine may
either be metabolized to cysteine by the transsulphuration
pathway or converted back to methionine by the remethy-
lation pathway [16,19,20]. Mutations in genes responsible
for homocysteine metabolism can result in homocystinuria,
a severe form of HHcy [20]. The most common genetic
cause of homocystinuria, homozygous cystathionine ␤-syn-
thase (CBS) deficiency, results in plasma homocysteine
concentrations of up to 400 ␮mol/L, compared to normal
plasma levels of ⬃10 ␮mol/L [16,19,20]. Homozygous
CBS deficiency is inherited as an autosomal recessive dis-
order with pleiotropic clinical manifestations, including
mental retardation, ectopia lentis, osteoporosis, skeletal ab-
normalities and hepatic steatosis [16,20]. Furthermore, pa-
tients are at higher risk for premature atherosclerosis and
thrombotic disease, which is the major cause of death
[16,19,21–23]. While homozygous CBS deficiency is rare,
heterozygous CBS deficiency occurs in approximately 1%
of the general population and is associated with premature
atherosclerosis and thrombotic disease in phenotypically
normal individuals [13,15,16,21–23]. Deficiency of 5,10-
methylenetetrahydrofolate reductase (MTHFR), the enzyme
involved in folate-dependent remethylation of homocys-
teine to methionine, also causes severe HHcy and can lead
to premature atherosclerosis and thrombotic disease [24 –
26]. Additionally, nutritional deficiencies in other vitamin
cofactors required for homocysteine metabolism, namely
folate, vitamin B6 (pyridoxal phosphate), B12 (methylco-
balamin), and B2 (riboflavin) can promote HHcy [27–31].
It has been estimated that inadequate intake of B-vita-
mins and folate may account for approximately two thirds
of all cases of HHcy [28]. The decision of both American
and Canadian governments to enrich cereal grain flour with
folate in the late 1990s to prevent neural tube defects
(NTDs) was thus seen as having great potential in prevent-
ing HHcy in the general public. Cohort studies have since
showed that folate fortification of cereal flour products has
been effective in reducing total plasma homocysteine levels
in the general population [32–34]. It is not yet known,
however, if lowering of total plasma homocysteine via fo-
late supplementation is effective in reducing the risk of
developing atherosclerosis and thrombosis [32]. Prospective
studies are currently underway to examine this association.
Regardless of the underlying condition leading to HHcy, the
relationship between elevated plasma homocysteine levels
and vascular disease persists.
3. Homocysteine and vascular disease
Patients suffering from homocystinuria develop exten-
sive arterial intimal thickening and fibrous plaques rich in
smooth muscle cells and collagen [20,23,35]. These fibrous
lesions greatly outnumber fatty atherosclerotic lesions in the
major arteries of homocystinuric patients and, combined
with abnormally accelerated thrombosis, lead to tissue in-
farction and death at an early age [35]. Venous thrombosis
and, to a lesser extent, arterial thrombosis are common in
patients with homocystinuria [36]. Homocysteine-induced
thrombosis seems to result from disturbances in the throm-
botic potential of endothelial cells rather than changes in
platelet physiology. This leads to the development of a
prothrombotic phenotype consistent with vascular injury
(reviewed in [19,20]).
It is estimated that 5 to 7% of the general population has
mild to moderate HHcy [13–16] and are at an increased risk
of developing coronary atherosclerosis and vascular throm-
bosis in later life. Individuals with mild HHcy also have an
increased prevalence of carotid artery stenosis, premature
peripheral and cerebral vascular atherosclerosis [19 –21,
23]. Epidemiological data has generally showed a strong
association between plasma homocysteine and the prema-
ture development of atherosclerotic cardiovascular disease.
Many case-control and cross-sectional studies have identi-
fied an association between total plasma homocysteine lev-
els and the development of carotid, coronary, peripheral and
aortic atherosclerotic disease [37]. The multicentre Euro-
pean Concerted Action Project (750 patients, 800 controls),
for example, revealed that HHcy confers an independent
risk similar to smoking and hypercholesterolemia for devel-
oping occlusive arterial disease [38]. Additionally, this
study suggested that mild HHcy may have a synergistic
effect when combined with other risk factors, such as dia-
betes and hypertension. Control of mild HHcy may there-
fore be critically important in attenuating lesion formation
 A.B. Lawrence de Koning et al. / Clinical Biochemistry 36 (2003) 431– 441
and preventing atherosclerosis when other established risk
factors are present. Unlike case-control studies, however,
prospective cohort studies have not shown a robust or con-
sistent association between HHcy and atherosclerosis [39].
This seems to be especially true for prospective studies of
healthy individuals with no previous history of cardiovas-
cular disease. Contrasting findings from prospective studies
have been suggested to be a result of differing experimental
designs and endpoints or perhaps even genetic or nutritional
heterogeneity of test populations, resulting in different car-
diovascular risk profiles in the populations studied [40].
Despite these conflicting prospective studies, mild HHcy is
generally regarded as a causal, independent risk factor for
occlusive vascular disease [18,41,42].
4. Potential cellular mechanisms by which
homocysteine promotes atherosclerosis
4.1. Inflammatory response
In general, the development and progression of athero-
sclerosis is considered to be a form of chronic inflammation
[1–3,11]. In support of these findings, in vitro studies have
demonstrated that homocysteine enhances the production of
several pro-inflammatory cytokines. Expression of mono-
cyte chemoattractant protein 1 (MCP-1) is increased in
cultured human vascular endothelial cells, smooth muscle
cells and monocytes treated with homocysteine [43– 45].
MCP-1 is known to enhance monocyte endothelial binding
and recruitment to the subendothelial cell space, a critical
early step in the formation of fatty streaks [11]. Homocys-
teine has also been shown to increase expression of IL-8
[43], a T-lymphocyte and neutrophil chemoattractant, in
cultured endothelial cells. Homocysteine-induced expres-
sion of MCP-1 and IL-8 in monocytes and endothelial cells
has been shown to occur through activation of NF-␬B, a
transcription factor involved in mediating downstream in-
flammatory processes [46]. Active NF-␬B, which is found
in atherosclerotic lesions, stimulates production of cyto-
kines, chemokines, interferons, leukocyte adhesion mole-
cules, hemopoietic growth factors and major histocompati-
bility (MHC) class I molecules—all of which are thought to
influence vascular inflammation and, ultimately, atherogen-
esis [46]. Recent data substantiating the in vivo activation of
NF-␬B and downstream inflammatory marker expression in
atherosclerotic lesions in hyperhomocysteinemic apoli-
poprotein E (apoE)-deficient mice further supports the con-
cept that homocysteine contributes to the development of
atherosclerosis by causing vascular inflammation [47].
4.2. Oxidative stress
Homocysteine is injurious to vascular endothelial cells
and impairs normal cellular function. One of the most rec-
ognizable physiological changes associated with endothelial
433
damage by HHcy is the impairment of vasodilation [48 –
52]. Abnormal vasomotor response is believed to be an
early step in the formation of atherosclerotic lesions [53].
Mild to moderate HHcy in both human patients and animal
models has been shown to impair normal endothelium-
dependent vasodilation [49,51,54]. B vitamin supplementa-
tion, which has been shown to lower plasma homocysteine
levels, is effective in restoring normal endothelial function
[55]. The additional observation that antioxidants prevent
impairment in endothelium-dependent relaxation suggests
the involvement of reactive oxygen species (ROS) [47–54].
The thiol group of homocysteine readily undergoes auto-
oxidation in plasma to generate ROS, and it has been sug-
gested that homocysteine induces cell injury/dysfunction
via a mechanism involving oxidative stress [50,56]. How-
ever, this hypothesis fails to explain why cysteine, which is
present in plasma at 20 to 30 fold higher concentrations than
homocysteine and is more readily auto-oxidized, does not
cause endothelial cell injury and is not considered a risk
factor for cardiovascular disease [57–59]. Recent studies
have also demonstrated that homocysteine does not signif-
icantly increase the production of ROS via auto-oxidation
and is largely involved in antioxidant and reductive cellular
biochemistry [60]. Based on these findings, a re-examina-
tion of the role of oxidative stress due to the auto-oxidation
of homocysteine is warranted.
Homocysteine-induced oxidative stress may occur
through other mechanisms. Various ex vivo studies using
vascular tissues have implicated HHcy in causing abnormal
vascular relaxation responses by enhancing the intracellular
production of superoxide anion (O2⫺) [53,61,62]. O2⫺ is
believed to react with and decrease the availability of en-
dothelial nitric oxide (NO) to yield peroxynitrite, thereby
limiting normal vasodilation responses [63,64]. O2⫺ and
peroxynitrite are also known to contribute to the oxidative
modification of tissues, resulting in the formation of lipid
peroxides and nitrosated end products such as 3-nitroty-
rosine. The observations that homocysteine decreases the
expression of a wide range of antioxidant enzymes [65,66]
and impairs endothelial NO bioavailability by inhibiting
glutathione peroxidase activity [52,67] raises the possibility
that homocysteine sensitizes cells to the cytotoxic effects of
agents or conditions known to generate ROS. Decreased NO
bioavailability has also been shown ex vivo to increase the
expression of MCP-1, which may enhance intravascular
monocyte recruitment and lead to accelerated lesion forma-
tion [68].
4.3. Endoplasmic reticulum stress
In eukaryotic cells, the endoplasmic reticulum (ER) is
the cellular organelle where secretory proteins or proteins
destined for the plasma membrane undergo a variety of
modifications, including disulphide bond formation, glyco-
sylation, folding and oligomeric assembly (see reviews
[69 –71]). To assist in the correct folding of newly synthe-
434 A.B. Lawrence de Koning et al. / Clinical Biochemistry 36 (2003) 431– 441
Fig. 2. Proximal sensors IRE-1, ATF-6 and PERK co-ordinately regulate
UPR signaling in eukaryotes. ER transmembrane protein IRE-1 dimerizes
and autophosphorylates upon release of GRP78 to bind unfolded proteins.
Endoribonuclease activity of IRE-1 cleaves mammalian XBP-1 mRNA to
remove a 26-base pair intron. Translation of alternatively spliced XBP-1
mRNA yields XBP-1 protein containing a novel C-terminus that binds to
ER stress response element (ERSE) promoters, increasing transcription of
ER chaperone genes. ATF-6 migrates to the Golgi following GRP78
release and is cleaved by site 1 and site 2 (S1/S2) proteases. The released
50 to 60 kDa ATF-6 transactivation domain migrates to the nucleus and
binds to ERSE, increasing transcription of ER chaperones. PERK is acti-
vated via dimerization and autophosphorylation following GRP78 release.
PERK catalyzes the phosphorylation of eukaryotic initiation factor-2␣
(eIF-2␣), which prevents eIF-2␣ from initiating translation, thereby limit-
ing the unfolded protein load on the ER. Activation of the PERK pathway
has also been shown to mediate the transcriptional activation of ER stress
response genes.
sized proteins and to prevent aggregation of folding inter-
mediates, the ER contains a high concentration of molecular
chaperones including the 78-kDa glucose-regulated protein
(GRP78), GRP94, calnexin, calreticulin, protein disulphide
isomerase and ERp72. These chaperones act as a quality
control system by ensuring that only correctly folded pro-
teins are allowed to enter the Golgi for further processing
and secretion. The observation that these chaperones are
induced in response to a variety of pathological agents
and/or conditions that cause ER stress [70] suggests that
they play a critical role in quality processes during protein
folding and processing.
ER stress results in the activation of the unfolded protein
response (UPR), an intracellular signaling pathway essential
for survival of cells undergoing ER stress (Figure 2). In
mammalian cells, the UPR is mediated via three ER-resi-
dent sensors: a type-I ER transmembrane protein kinase
(IRE-1), the activating transcription factor 6 (ATF-6) and
the PKR like ER kinase (PERK). Activation of these three
pathways is mediated by GRP78, which is bound to each
sensor in the absence of ER stress. During the accumulation
of unfolded proteins in the ER, GRP78 dissociates from its
respective sensor to interact with unfolded proteins, thereby
facilitating the activation of IRE-1, ATF-6 and PERK [72–
74]. Activation of both IRE-1 and ATF-6 increase the ex-
pression of ER-resident chaperones. IRE-1 is a stress-acti-
vated transmembrane protein kinase having
endoribonuclease activity. Following ER stress, IRE-1
dimerizes and is autophosphorylated, thereby allowing
IRE-1 to act as an endoribonuclease in the alternative splic-
ing of XBP-1 mRNA. The removal of a 26 base pair intron
results in a translation frameshift that permits XBP-1 to act
as a transcriptional activator of genes containing upstream
ER stress response elements (ERSE). ATF-6 is a transcrip-
tion factor that is localized to the ER membrane in un-
stressed cells. Upon ER stress, ATF-6 is transported to the
Golgi where the cytosolic transactivation domain of ATF-6
is cleaved from the membrane by specific proteases (S1P
and S2P) that also recognize and cleave sterol regulatory
element-binding proteins (SREBPs) under conditions of ste-
rol starvation. Following release, the transactivation domain
of ATF-6 localizes to the nucleus where it binds to ERSE,
thereby activating transcription of numerous UPR-respon-
sive genes, including GRP78, GADD153 and ERp72. Con-
comitant with an increase in the transcriptional activation of
UPR-responsive genes, ER stress also leads to a rapid,
general decrease in protein synthesis, a cellular process
mediated by the transmembrane protein kinase, PERK. Ac-
tivation of PERK causes phosphorylation of eukaryotic ini-
tiation factor-2␣ (eIF-2␣), which effectively blocks mRNA
translation initiation to help relieve the unfolded protein
burden on the ER. Recent studies have also demonstrated
that PERK-dependent eIF-2␣ phosphorylation is required
for transcriptional activation of a wide range of UPR-re-
sponsive genes [75,76]. As a result, the UPR co-ordinately
enhances cell survival by ensuring that the adverse effects
of ER stress are dealt with in a timely and efficient manner.
Failure to elicit a functional UPR following ER stress can
result in programmed cell death via specific ER-dependent
pathways and contribute to the pathogenesis of a number of
human diseases, including diabetes, Alzheimer’s disease,
Parkinson’s disease and cancer [71].
A recently proposed mechanism of vascular injury in-
volves the ability of homocysteine to cause ER stress by
disrupting disulphide bond formation and causing misfold-
ing of proteins traversing the ER [65,66,77–79]. We as well
as others have previously reported that elevated levels of
intracellular homocysteine increase the expression of sev-
eral ER stress response genes, including GRP78, GRP94,
Herp and RTP [65,66,77,78,80]. In addition, homocysteine
induces the expression of GADD153 [65,66,80], a basic
region leucine zipper transcription factor involved in ER
stress-induced cell death [81]. These effects in gene expres-
sion directly involve the UPR because homocysteine treat-
ment has been shown to induce the activation of both PERK
[82,83] and IRE-1 [84,85].
Transient and chronic ER stress elicited by homocysteine
has been shown to adversely affect several cellular func-
tions involved in the development and progression of ath-
 A.B. Lawrence de Koning et al. / Clinical Biochemistry 36 (2003) 431– 441
erosclerosis, including lipid dysregulation, programmed cell
death and inflammation. We have recently demonstrated
that homocysteine-induced ER stress causes dysregulation
of lipid biosynthesis by activating the SREBPs [80], ER-
resident transcription factors responsible for the induction
of genes in the cholesterol/triglyceride biosynthesis and
uptake pathways [86,87]. The observation that stable over-
expression of GRP78, which protects cells from agents
and/or conditions known to cause ER stress, inhibits homo-
cysteine-induced cholesterol gene expression in cultured
human cells further supports an association between ER
stress and lipid metabolism [80]. Additional studies have
demonstrated that activation of the UPR increases lipid
biosynthesis in yeast [88,89] and dolichol biosynthesis [90],
which is a component of the cholesterol biosynthesis path-
way in cultured mammalian cells. Our findings suggest that
activation of the UPR by homocysteine promotes the over-
production of ER lipid components, including cholesterol
and triglycerides, resulting in the accumulation of lipids in
affected cell types, including hepatocytes, smooth muscle
cells and macrophages. This dysregulation in lipid biosyn-
thesis and uptake may explain the paradox as to why lipid-
rich atherosclerotic lesions develop in hyperhomocysteine-
mic patients with normal serum lipid profiles.
Recent studies have now shown that homocysteine in-
duces programmed cell death in cultured human vascular
endothelial cells and that this effect involves activation of
the UPR [85]. Homocysteine-induced cell death was mim-
icked by other ER stress agents and was dependent on
IRE-1 signaling. Activation of IRE-1 by homocysteine
leads to a rapid and sustained activation of JNK protein
kinases [91], a result consistent with the finding that acti-
vation of JNK by ER stress involves binding of IRE-1 to
TRAF2 [92]. Because persistent activation of JNK corre-
lates with cell death [93], these studies provide further
support for a mechanism involving homocysteine-induced
programmed cell death. In addition, caspase-3 or a caspase-
like protease was shown to be essential for homocysteine-
induced programmed cell death, a result consistent with the
ability of homocysteine thiolactone, a cyclic thioester de-
rivative of homocysteine synthesized by specific aminoacyl-
tRNA synthetases [94], to induce programmed cell death in
HL-60 cells [95]. Although caspase-7 mediated caspase-12
activation has been implicated in the coupling of ER stress
to programmed cell death [96], there are presently no re-
ported studies examining the effect of homocysteine on the
activation of these caspases. Given that programmed cell
death has been widely documented to occur in animal and
human atherosclerotic lesions, and that programmed cell
death and ER stress are increased in atherosclerotic lesions
from mice fed hyperhomocysteinemic diets (Zhou and Aus-
tin, unpublished results), it is tempting to suggest that ho-
mocysteine could adversely affect the stability and/or
thrombogenicity of atherosclerotic lesions through its abil-
ity to elicit ER stress. Further studies are, however, required
to identify the pro-apoptotic factors involved in this process
435
as well as their in vivo relevance to the development and
progression of atherosclerosis.
Previous work by Pahl and colleagues [97–100] has
demonstrated that the in vitro activation of NF-␬B occurs in
cells undergoing ER stress. This suggests that homocys-
teine-induced ER stress could contribute to the development
of atherosclerosis by directly enhancing an inflammatory
response via NF-␬B activation. Recent studies have now
demonstrated that HHcy enhances activation of NF-␬B in
atherosclerotic lesions and in selective tissues from apoE-
deficient mice having diet-induced HHcy [47]. Although the
mechanism responsible for this proinflammatory effect of
HHcy is unknown, homocysteine-induced ER stress could
enhance the phosphorylation and activation of JNK, thereby
leading to further inflammation and programmed cell death
[92]. Based on these recent findings, homocysteine-induced
ER stress may prove to be an important mechanism that not
only contributes to the development of atherosclerosis but
also influences other specific diseases, including diabetes
and hypertension [71].
4.4. Additional mechanisms
In addition to the established mechanisms described
above, other studies have suggested that elevated levels of
homocysteine can elicit other effects that contribute to
atherogenesis and thrombotic disease. It has been reported
that homocysteine stimulates the proliferation of cultured
vascular smooth muscle cells [101–103] and upregulates
smooth muscle cell collagen [104], findings consistent with
the observation that patients with severe HHcy have wide-
spread premature atherosclerosis with intimal thickening
and collagen-rich, fibrous lesions [15]. Several potential
mechanisms have also been proposed to explain endothelial
cell dysfunction during HHcy, including direct cell injury
[50,56], growth arrest [65] and altered cellular methylation
status [105]. In addition to causing endothelial cell dysfunc-
tion, elevated levels of homocysteine have been shown to
increase the procoagulant activity of cultured endothelial
cells by enhancing tissue factor [106 –108] and factor V
[109] activities, inhibiting cell surface thrombomodulin
[110] and protein C [111], and impairing tissue plasmino-
gen activator binding to its endothelial cell receptor, an-
nexin II [112].
Homocysteine thiolactone, the cyclic thioester of homo-
cysteine, has recently received attention for its potential role
in atherosclerosis and thrombotic disease [94,113]. In addi-
tion to the transsulphuration and remethylation pathways,
intracellular homocysteine can be converted by methionyl-
tRNA synthase into a homocysteine-AMP complex. This
complex is subsequently catabolised to homocysteine thio-
lactone, thereby preventing the incorporation of homocys-
teine into nascent polypeptide chains. However, homocys-
teine thiolactone has unique reactive properties that can lead
to the homocysteinylation of lysine residues and free amine
groups on numerous cellular proteins, thereby resulting in
436 A.B. Lawrence de Koning et al. / Clinical Biochemistry 36 (2003) 431– 441
decreased biological activity and premature degradation
[113]. In addition, homocysteine thiolactone secreted into
the circulation may induce widespread modification of
plasma proteins that could potentially contribute to the de-
velopment of atherosclerosis and thrombotic disease. Re-
cent studies have demonstrated that homocysteine thiolac-
tone decreases paraoxonase activity associated with HDL,
thereby rendering HDL less protective against oxidative
damage and against toxicity of homocysteine thiolactone
[114]. Additional studies using physiologically relevant in
vivo models of HHcy are necessary to further understand the
atherogenic role of homocysteine thiolactone.
5. Animal models of hyperhomocysteinemia
Recent findings from genetic- and diet-induced animal
models of HHcy have significantly enhanced the status of
homocysteine as a risk factor for atherosclerosis. They have
also supported and extended many of the proposed in vitro
mechanisms and have provided a more physiological per-
spective on the role of homocysteine in the induction of
cardiovascular disease.
Manipulation of plasma homocysteine can be accom-
plished by dietary and/or genetic approaches. The addition
of methionine and/or depletion of B vitamins and folate in
the diet can be used to induce mild to severe HHcy. Genet-
ically engineered mice deficient in CBS or MTHFR have
also been used as animal models of HHcy. Homozygous
CBS-deficient mice have total plasma homocysteine levels
40 times greater than normal and suffer from severe growth
retardation, hepatic steatosis and ectopia lentis – phenotypic
changes also observed in patients with homozygous CBS
deficiency [115]. Heterozygous CBS-deficient mice, how-
ever, do not suffer from the same developmental defects as
homozygotes. These animals have twice the normal concen-
tration of plasma homocysteine, making them ideal models
to study mild HHcy. Recently, the chronic, mild HHcy
present in heterozygous CBS-deficient mice has been shown
to cause endothelial dysfunction by decreasing vascular NO
bioavailability, thereby leading to impaired vasorelaxation
[48,52]. This association is not unique to CBS-deficient
mice, as endothelial dysfunction has also been shown in
rabbits and cynomolgus monkeys (Macaca fascicularis)
having diet-induced HHcy [53,116 –118]. Although CBS-
deficient mice and nonmurine models of HHcy exhibit en-
dothelial dysfunction, it is important to note that they do not
develop atherosclerosis [115–118].
MTHFR-deficient mice have been recently developed to
examine the effects of HHcy resulting from genetic defi-
ciencies in the remethylation pathway [119]. Although
MTHFR-deficient mice share basic phenotypic similarities
with CBS-deficient mice, they are unique in ways that may
predispose them to developing atherosclerosis. Mature het-
erozygous and homozygous MTHFR-deficient mice accu-
mulate lipid in their proximal aorta which is reminiscent of
early atherosclerotic lesions [119]. This is significant, given
that plasma lipid levels are normal in these animals. Al-
though the arterial lipid inclusions are not as elaborate as
lesions observed in murine models of atherosclerosis, such
as apoE-deficient or LDL receptor-deficient mice, they are
the first observations showing that mild HHcy is involved in
the formation of vascular lesions.
Studies in our laboratory examined the molecular and
cellular consequences of diet-induced HHcy in wild-type
and heterozygous CBS-deficient mice [80]. Both mild and
severe HHcy were shown to induce ER stress and abnormal
hepatic accumulation of lipid in each strain of mice. ER
stress was shown to promote SREBP activation, thereby
resulting in increased expression of enzymes encoding
genes for cholesterol and triglyceride biosynthesis, as well
as the LDL receptor. SREBP dysregulation resulted in he-
patic steatosis, a characteristic feature observed in human
homocystinuric patients. Interestingly, plasma lipid levels
remained unchanged despite increases in hepatic VLDL-
triglyceride secretion rates. These observations suggest that
hepatic steatosis in HHcy does not result from impaired
lipid export, but instead is due to increased lipid biosynthe-
sis coupled with enhanced uptake of LDL by the liver.
Should this effect of homocysteine on lipid metabolism and
uptake be confirmed in vascular cells involved in athero-
genesis, this could potentially explain the lipid rich arterial
lesions associated with mild HHcy in patients with normal
lipid profiles.
One of the earliest detectable cellular responses in the
development of atherosclerotic lesions is the binding of
monocytic cells to the vascular endothelium. Recent studies
have now demonstrated that the number of monocytes
present on the surface of aortic endothelium is significantly
elevated in rats with diet-induced HHcy, although typical
atherosclerotic lesions were not observed [120]. A signifi-
cant increase in the expression of MCP-1, VCAM-1 and
E-selectin was also observed in the aortic endothelium of
hyperhomocysteinemic rats, thereby providing a potential
cellular mechanism responsible for the increase in mono-
cyte binding and recruitment. The additional observation
that folate supplementation prevented an increase in plasma
homocysteine levels, decreased monocyte binding to the
endothelium and inhibited the expression of MCP-1,
VCAM-1 and E-selectin, emphasizes a potential antiathero-
genic effect of lowering plasma homocysteine.
Since the development and progression of atherosclero-
sis is limited in some animal models of HHcy, we and other
researchers have generated dietary HHcy in genetically al-
tered mice prone to developing atherosclerosis. An estab-
lished model of spontaneous atherosclerosis is the apoE-
deficient mouse [121]. These mice develop
hypercholesterolemia because of impaired reverse choles-
terol transport to the liver, thereby leading to the accumu-
lation of cholesterol-rich remnants in plasma. This persis-
tent and severe hypercholesterolemia (up to 5 times normal)
leads to the premature development of complex atheroscle-
 A.B. Lawrence de Koning et al. / Clinical Biochemistry 36 (2003) 431– 441
Fig. 3. Atherosclerotic lesions in the aortic root of mice fed control (A),
high homocysteine (B) or high methionine diet (C). Lesion sizes in the
hyperhomocysteinemic mice (B, C) were significantly larger, compared to
the control mice (A). Atrophy of the tunica media and rupture of the elastic
laminae were often observed (arrow in B). All sections were stained with
Orcein to reveal the elastic lamina. Data were kindly provided by Drs. Ji
Zhou and Erling Falk.
rotic lesions [121]. Two separate studies published in the
last two years demonstrate that mild HHcy accelerates
atherogenesis in apoE-deficient mice. Zhou et al. [122]
showed that apoE-deficient mice fed methionine- or homo-
cysteine-enriched diets developed larger, more complex and
more numerous arterial lesions, compared to mice fed con-
trol diets (Figure 3). An important feature of these patho-
logical changes was that they occurred without a significant
elevation in the concentration of plasma lipids. Many of
these lesions were rich in collagen, probably owing to the
ability of homocysteine to stimulate SMC proliferation and
extracellular matrix deposition [104,123]. Zhou et al. fur-
ther demonstrated that the differences in lesion size and
frequency were seen only between groups following 3
months of dietary treatment compared to 12 months, sug-
gesting that HHcy mediates the early stages of atherogene-
sis [122]. In a separate study using apoE-deficient mice fed
diets supplemented with methionine to induce HHcy, Hof-
mann et al. [47] found significant increases in atheroscle-
rotic lesion area, compared to mice fed control diets. Fur-
thermore, diet-induced HHcy promoted NF-␬B activation in
aortic tissue, leading to the increased expression of the
pro-inflammatory adhesion molecule VCAM-1 as well as
the pro-inflammatory mediators RAGE and EN-RAGE. In-
creased expression of pro-inflammatory genes in the aorta
of hyperhomocysteinemic animals is also consistent with
the finding of increased plasma concentrations of TNF-␣
and increased numbers of lesion-resident macrophages. As
discussed previously, the ability of homocysteine to activate
437
NF-␬B and enhance vascular inflammatory processes may
in part be related to its ability to cause ER stress [97–99].
Our finding that markers of ER stress are increased in the
livers [80] and atherosclerotic lesions (Zhou and Austin,
unpublished results) of mice with diet-induced HHcy pro-
vides support for this concept. Lesions with increased ex-
pression of the collagen degrading matrix metalloprotein-
ase-9 were also observed, which could potentially promote
lesion instability and rupture. The increased collagen in the
lesions observed by Zhou et al. [122] is in contrast to the
prediction of plaque instability by Hofmann et al. [47]. Both
studies, however, are in agreement that diet induced HHcy
in the presence of a hyperlipidemic state accelerates the
development of atherosclerosis. A recent study by Wang et
al. [124], using apoE and CBS double knockout mice,
showed that atherosclerotic lesion area and lesion lipid
content increased with plasma homocysteine concentra-
tions, independent of diet. Overall, these data strongly sug-
gest that HHcy amplifies proatherogenic cellular processes
when combined with hyperlipidemia. As previously men-
tioned, case-control epidemiological studies seem to impli-
cate HHcy in compounding the overall risk of cardiovascu-
lar disease when in the presence of additional risk factors
such as hypertension and diabetes [35,37,38]. Supporting
this hypothesis, Matthias et al. [125] showed that sponta-
neously hypertensive rats fed high doses of methionine
developed more complex arterial lesions compared to rats
fed normal diets.
Collectively, these studies have helped elucidate the
causal role of homocysteine in the development of endothe-
lial dysfunction and atherosclerosis. Furthermore, these an-
imal models are valuable in vivo tools to further examine
potential therapeutic approaches in lowering plasma homo-
cysteine while decreasing the prevalence of cardiovascular
disease.
6. Conclusions and questions
HHcy is an independent risk factor for cardiovascular
disease. Recent studies have identified three major cellular
mechanisms by which HHcy may contribute to the devel-
opment of endothelial dysfunction and atherosclerosis.
They include the induction of pro-inflammatory factors,
oxidative stress, and ER stress. Genetic- and diet-induced
animal models of HHcy have now demonstrated a causal
relationship between HHcy and accelerated atherosclerosis.
They have also provided important insights into the role of
HHcy in endothelial dysfunction and the development of
atherosclerosis. These studies, however, raise some inter-
esting and relevant questions. Although HHcy seems to be
very important in early lesion development, what role does
it play in plaque stability and/or thrombogenicity? Can
HHcy, in the absence of hypercholesterolemia or other sig-
nificant risk factors, enhance lesion development in hu-
mans? Does dietary enrichment of vitamins essential for the
438 A.B. Lawrence de Koning et al. / Clinical Biochemistry 36 (2003) 431– 441
metabolism of homocysteine protect against cardiovascular
disease? Answering these and other important questions
will certainly enhance our understanding of the role of
HHcy in the development and progression of atherosclero-
sis.
Acknowledgments
The author’s work is supported by Research Grants from
the Heart and Stroke Foundation of Ontario (T-4005) and
the Canadian Institutes of Health Research (MOP-49417).
R.C. Austin is a Career Investigator of the Heart and Stroke
Foundation of Ontario. We apologize to many of our col-
leagues whose work could not be directly acknowledged
due to space limitations.
Note added to proof
Recent studies by Ji and Kaplowitz [126] have demon-
strated increased HHcy, ER stress and liver injury in an
alcohol-fed mouse model of HHcy.
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