Atherosclerosis 150 2000 265 – 274
The effect of carotenoids on the expression of cell surface adhesion molecules and binding of monocytes to human aortic
endothelial cells
K.R. Martin
1
, D. Wu, M. Meydani
Vascular Biology Program, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts Uni6ersity,
711
Washington Street, Boston, MA
02111
, USA Received 19 November 1998; received in revised form 4 June 1999; accepted 20 August 1999
Abstract
Several large epidemiological studies have shown a correlation between elevated plasma carotenoid levels and decreased risk of cardiovascular disease CVD. One proposed mechanism for the beneficial effect of carotenoids is through functional modulation
of potentially atherogenic processes associated with the vascular endothelium. To test this, we incubated confluent human aortic endothelial cell HAEC cultures passages 4 – 8 for 24 h with each of the five most prevalent carotenoids in human plasma, which
are a-carotene, b-carotene, b-cryptoxanthin, lutein, and lycopene, at an approximate concentration of 1 mmoll. Carotenoids were solubilized in 0.7 vv tetrahydrofuran and incorporated into FBS before adding to cell culture medium. Due to disparate
solubilities in aqueous medium, final concentrations of a-carotene, b-carotene, b-cryptoxanthin, lutein, and lycopene were 1.7, 1.1, 0.7, 0.9, and 0.3 mmoll and monolayers accumulated 647, 158, 7, 113, and 9 pmolmg protein, respectively. Monolayers were then
stimulated with IL-1b 5 ngml for 6 h with subsequent determination of cell surface expression of adhesion molecules as measured by an enzyme-linked immunosorbent assay ELISA. To assess endothelial cell adhesion to monocytes, IL-1b-stimulated
monolayers were incubated for 10 min with
51
Cr-labeled U937 monocytic cells and adhesion determined by isotope counting. Pre-incubation of HAEC with b-carotene, lutein and lycopene significantly reduced VCAM-1 expression by 29, 28, and 13,
respectively. Pre-incubation with b-carotene and lutein significantly reduced E-selectin expression by 38 and 34, respectively. Pre-treatment with b-carotene, lutein and lycopene significantly reduced the expression of ICAM-1 by 11, 14, and 18,
respectively. While other carotenoids were ineffective, lycopene attenuated both IL-1b-stimulated and spontaneous HAEC adhesion to U937 monocytic cells by 20 and 25, respectively. Thus, among the carotenoids, lycopene appears to be most
effective in reducing both HAEC adhesion to monocytes and expression of adhesion molecules on the cell surface. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Carotenoids; Lycopene; Adhesion molecule; Endothelial cell; U937 www.elsevier.comlocateatherosclerosis
1. Introduction
Cardiovascular disease CVD remains a leading cause of morbidity and mortality in the United States
and atherosclerosis is a key factor in the pathogenesis of myocardial and cerebral infarction, gangrene, and
loss of function in the extremities [1]. Although numer- ous risk factors contribute to CVD, their presence is
only partially responsible for the incidence of CVD. A growing body of literature supports the hypothesis that
increased consumption of fruits and vegetables may substantially reduce the incidence of atherogenesis and
alleviate its underlying pathologic condition [2,3]. Re- sults of epidemiological studies are generally consistent,
showing a protective association between plant-derived antioxidants, such as carotenoids, and CVD [4,5].
Carotenoids are a family of naturally occurring plant pigments that were initially studied due to their role as
retinoid precursors. However, carotenoids have now been shown to function in immunomodulation, gap
Corresponding author. Tel.: + 1-617-5563126; fax: + 1-617- 5563344.
E-mail address
:
meydani –vblhnrc.tufts.edu M. Meydani
1
Present address: Laboratory of Transgenic Carcinogenesis, Na- tional Institute of Environmental Health Sciences, National Institutes
of Health, Research Triangle Park, NC. 0021-915000 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 2 1 - 9 1 5 0 9 9 0 0 3 7 5 - 5
junction communication, induction of carcinogen-me- tabolizing enzymes, and photoprotection, in addition to
their antioxidant properties [6]. More than 600 distinct carotenoids have been isolated from natural sources
and among these approxim. 50 are components of the US diet, with identification of 21 carotenoids in human
plasma [7]. There are five well-characterized carotenoids in most blood samples comprising approximately 90
of the total plasma pool of carotenoids. These are a-carotene, b-carotene, b-cryptoxanthin, lutein and ly-
copene [8].
Regulated expression of numerous cell surface adhe- sion molecules is a critical event in the binding of
normally non-thrombogenic circulating leukocytes such as the monocyte to the aortic endothelial surface and is
one of the earliest detectable events in human and experimental atherosclerosis [9,10]. The subsequent
transendothelial migration of these adherent leukocytes, their accumulation in the aortic intima, transformation
of monocytes into lipid-engorged foam cells, and secre- tion of cytokines and growth factors are important
events in the initiation and progression of atheroscle- rotic plaques [11]. Cell surface adhesion molecules are
important regulators of direct cell – cell interactions; inflammatory responses in atherogenesis are directed by
regulation and expression of these molecules [12]. Ad- hesion molecule expression in low-density lipoproteins
LDL receptor-knockout mice fed atherogenic diets showed a significantly reduced incidence of fatty streaks
supporting a role for adhesion molecules in atherogene- sis [13]. Constituents of each of the main families of
adhesion molecules are involved in the interactions of endothelial cells EC and immune cells. Intercellular
adhesion molecule ICAM-1 and -2 CD54 and CD102 and vascular cell adhesion molecule VCAM-
1, CD106 are members of the immunoglobulin super- family expressed on EC. Of these, ICAM-1 and
VCAM-1 increase in response to various inflammatory cytokines. E-Selectin and P-selectin CD62E and
CD62P on EC also play an early role in adhesion between these two cell types. ICAM-1 and -2 bind to
the LFA-1 counterligand; VCAM-1 binds to VLA-4; the selectins recognize certain carbohydrate structures
on opposing cells [14,15].
Oxidative stress
and expression
of adhesion
molecules on vascular EC are considered to be impor- tant features in the pathogenesis of atherosclerosis and
other inflammatory diseases [16,17]. Studies suggest a molecular linkage between an antioxidant-sensitive
transcriptional regulatory mechanism and expression of adhesion molecule genes that expands on the notion of
oxidative stress as an important regulatory signal in the pathogenesis of atherosclerosis [18]. In the inflamma-
tory response of the arterial wall, leukocyte recruitment to the endothelium is mediated by the interaction of
adhesion molecule receptors expressed on the surface of EC and immune cells.
While the pathogenic oxidation of LDL is currently one
postulated mechanism
for the
etiology of
atherosclerosis, the role of dietary antioxidants in sup- pressing the deleterious oxidation of LDL has produced
conflicting results, suggesting that other factors and possibly alternate mechanisms are involved. The spe-
cific contribution of carotenoids in the atherosclerosis process is inconclusive since carotenoids have been
shown to protect LDL in some studies but not in others. In fact, the extent of in vitro LDL oxidation
and protection by an antioxidant may not indicate in vivo atherosclerotic status as well as was recently em-
phasized [19]. However, in one study b-carotene supple- mentation in rabbits retarded aortic lesion formation
[20]. Also, in a more recent study b-carotene supple- mentation in combination with vitamins E and C re-
duced
aortic valve
lesion formation
in LDL
receptor-knockout mice [21], which may indicate a po- tential effect by carotenoids in the final pathological
outcome. Our contention is that carotenoids may act via alternate mechanisms within the endothelial cell to
modulate adhesion molecule expression and thus reduce subsequent leukocyte binding. As a result, the focus of
this research was to examine in vitro the effect of the five most prevalent plasma carotenoids on expression of
key adhesion molecules involved in the atherosclerosis process, and determine the subsequent binding of U937
monocytic cells when carotenoids are incorporated into human aortic endothelial cells HAEC. Our results
indicate that among the test carotenoids, lycopene ap- pears to be the most effective in reducing immune and
endothelial cell interaction.
2. Materials and methods
