Influence of homocysteine on the interaction between circulating monocytes and endothelial cells

Mild hyperhomocysteinemia is an independent risk factor for the
development of coronary artery disease, cerebrovascular disease and
peripheral arterial disease. The mechanisms by which
hyperhomocysteinemia promotes vascular disease are not completely
understood yet. An increasing body of evidence has implicated
oxidative stress as being contributory to homocysteine’s
deleterious effects on the vasculature. Elevated levels of
homocysteine lead to increased generation of superoxide anion in
endothelial cells by a biochemical mechanism involving nitric oxide
synthase, and, to a lesser extent, by an increase in the chemical
oxidation rate of homocysteine and other aminothiols in the
circulation. Furthermore, homocysteine has been shown to inhibit
the activity of important cellular antioxidant enzymes, like the
cellular isoform of glutathione peroxidase or superoxide dismutase,
which may contribute to homocysteine’s induced oxidant stress. The
resulting increase in reactive oxygen species leads to decreased
bioavailability of the endothelium-derived signaling molecule
nitric oxide via oxidative inactivation and thereby induces
endothelial dysfunction. This seems to play a central role in the
molecular mechanisms underlying the effects of homocysteine on
vascular function. Hyperhomocysteinemia not only leads to
endothelial dysfunction but also promotes the development and
propagation of atherosclerotic lesion in atherosclerosis-prone
animal models. As the recruitment of circulating monocytes to the
vessel wall plays a crucial role in the process of atherosclerosis,
the purpose of this study was to examine the influence of
homocysteine on the interaction of endothelial cells with
monocytes. Exposure of endothelial monolayers to D,L- and
L-homocysteine resulted in a time- and dose-dependent increase in
adherent THP-1 cells by upregulating ICAM-1 expression on
endothelial cells. L-cysteine and D-homocysteine had no effects.
This indicates that the stimulatory effect is specific for the
naturally occurring L-stereoisomer and rather a biochemical than a
chemical effect. The increased endothelial expression of ICAM-1
seems to be mediated by increased activation of the nuclear
transcription factor NF-kB, as shown by increased nuclear
translocation of NF-kB in homocysteine-incubated endothelial cells.
In accordance, inhibition of NF-kB translocation by a synthetic
inhibitor Bay 11-7082 significantly diminished homocysteine-induced
ICAM-1 expression and adhesion of monocytes to endothelial cells.
In addition, incubation of monocytes with D,L- homocysteine and
L-homocysteine resulted in significant increase in the number of
adhering monocytes to unstimulated endothelial monolayer by
upregulating the expression of beta-2 integrins. Furthermore,
homocysteine-incubation of endothelial cells and monocytes resulted
in a dose-dependent and significant increase in the intracellular
generation of reactive oxygen species. In support of the role of
increased oxidant stress for the above mentioned effects, treatment
of endothelial cells with the superoxide scavengers MnTBAP or Tiron
together with homocysteine abolished homocysteine-induced monocyte
adhesion, ICAM-1 expression and the nuclear translocation of NF-kB.
Incubation of THP-1 monocytes with Tiron abolished
homocysteine-induced beta-2 integrin expression on these cells and
adhesion to unstimulated endothelial cells. These findings suggest
that superoxide anion radicals mediate homocysteine’s effects on
endothelium-monocyte interactions. In addition to previous studies
that indicated that a significant source of reactive oxygen species
in homocysteine-treated endothelial cells might be endothelial
nitric oxide synthase, experiments using inhibitors of nitric oxide
synthase in THP-1 cells indicated that nitric oxide
synthase-dependent generation of superoxide anion also occurs in
homocysteine-incubated THP-1 cells. This mechanism may contribute
to homocysteine-induced oxidant stress. The information generated
from these studies may be helpful in designing intervention
strategies aimed at inhibiting the generation of reactive oxygen
species in the vasculature that is associated with signaling events
of monocyte recruitment and infiltration involved in
atherosclerosis.