Trans-fatty acids induce pro-inflammatory responses and endothelial
Kevin A. Harvey1, Tyler Arnold1, Tamkeen Rasool1, Caryl Antalis1, Steven J. Miller2
and Rafat A. Siddiqui1,3,4*
1Cellular Biochemistry Laboratory, Methodist Research Institute, Clarian Health,
1701 N. Senate – Room E504, Indianapolis, IN 46202, USA
2Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA
3Department of Biology, Indiana University-Purdue University, Indianapolis, IN, USA
4Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA
(Received 19 January 2007 – Revised 13 July 2007 – Accepted 16 July 2007)
Epidemiological data indicate that there is a strong association between intake of trans-18:2 fatty acids (TFA) and sudden cardiac death. There is
little known about the mechanisms by which TFA exert harmful effects on the cardiovascular system. The present in vitro study is the first to
demonstrate the effects of membrane-incorporated C18:2 TFA on human aortic endothelial cell (HAEC) function. Trans-18:2 fatty acids
were incorporated to a greater extent (2-fold) in the phospholipid fraction of endothelial cells than that of cis-18:2; furthermore, these fatty
acids were enriched to a similar extent in the TAG fraction. Flow cytometric analysis indicated that TFA treatment of HAEC significantly
increased the expression of endothelial adhesion molecules, including intercellular adhesion molecule-1 (CD54) and vitronectin receptor
(CD51/CD61). Incorporation of TFA into membranes increased HAEC adhesion to fibronectin- or vitronectin-coated plates by 1·5- to 2-fold,
respectively. Neutrophil and monocyte adhesion to HAEC monolayers was nearly proportional to adhesion molecule expression. TFA treatment
also induced the release of monocyte chemoattractant protein-1 by nearly 3-fold in non-stimulated HAEC. Furthermore, we examined the role of
TFA on in vitro angiogenic assays. Chemotactic migration of TFA-treated HAEC toward sphingosine-1-phosphate (SPP) was significantly
increased compared with controls. Conversely, capillary morphogenesis of TFA-treated HAEC was significantly inhibited in response to SPP,
suggesting that TFA incorporation suppresses endothelial cell differentiation. In conclusion, these in vitro studies demonstrated that TFA play
a role in the induction of pro-inflammatory responses and endothelial cell dysfunction.
Endothelial cells: Trans-fatty acids: Adhesion molecules: Chemotaxis: Capillary morphogenesis
After the first successful hydrogenation of oils in 1897, the
proportional intake of trans isomers of unsaturated fatty
acids has dramatically risen in the human diet1. Trans-fatty
acid consumption is estimated to contribute 4–12% of the
total dietary fat intake in the US population, which corre-
sponds to 13g trans-fatty acids/person per d at the higher
intake2. Unlike Western diets, traditional diets in Korea and
Japan contain relatively small quantities of trans-fatty acids,
with estimates in the range of 0·1–0·6g/person per d3.
Trans-fatty acids occur naturally at relatively low levels in
meat and dairy products as a by-product of fermentation in
ruminant animals1. The majority of trans-fatty acids in the
diet are trans-8:1, which is derived from the partial hydrogen-
ation of oils4. However, the process of heating vegetable oils
during deodorisation, and frying or baking food in vegetable
oils results in the generation of trans-18:25. The elevated
temperature in these processes causes the conversion of cis
double bonds to trans isomers.
The effect of increased trans-fatty acid consumption has
been linked to a variety of afflictions, most notably CHD.
Numerous epidemiological studies have correlated elevated
dietary intake of trans-fatty acids with increased morbidity
and mortality from CHD. Willett suggested that replacing par-
tially hydrogenated fat with natural non-hydrogenated veg-
etable oils could prevent 30000–100000 CHD-related
premature deaths each year6. By evaluating fatty acid intake
and mortality over 25 years, the Seven Countries Study
reported a correlation between trans-fatty acid consumption
and the risk of death from CHD (r 0·78; P,0·001)7. Similar
findings were also reported in the Health Professionals
Cancer Prevention Study9and the Nurses’ Health Study10.
A Danish study also linked trans-fatty acid consumption to
the development of atherosclerosis11.
Compared with the consumption of an equal amount of
energy from saturated or cis-unsaturated fats, the consumption
*Corresponding author: Dr Rafat Siddiqui, fax þ1 317 962 9369, email email@example.com
Abbreviations: EBM-2, endothelial cell basal medium-2; HAEC, human aortic endothelial cells; MCP-1, monocyte chemoattractant protein-1; SPP, sphingosine-1-
British Journal of Nutrition (2008), 99, 723–731
q The Authors 2007
British Journal of Nutrition
of trans-fatty acids raises levels of LDL-cholesterol, reduces
levels of HDL-cholesterol and increases the total cholester-
ol:HDL-cholesterol ratio, a powerful predictor of the risk of
CHD12. Although these effects would be expected to increase
the risk of CHD, the relationship between the intake of trans-
fats and the incidence of CHD reported in prospective studies
has been greater than that predicted by changes in serum lipid
levels alone13–15, suggesting that trans-fatty acids may also
influence other risk factors for CHD.
Recent studies suggest multiple possible mechanisms that
might mediate the association of trans-fatty acids with
CVD16. For example, trans-fatty acids influence PG balance,
which in turn promotes thrombogenesis17and inhibits the con-
version of linoleic acid to arachidonic acid and to other n-6
PUFA, perturbing essential fatty acid metabolism and causing
changes in the phospholipid fatty acid composition in the
aorta18. Trans-fatty acids have been associated with the acti-
vation of systemic inflammatory responses, including substan-
tially increased levels of IL-6, TNF-a, TNF receptors and
monocyte chemoattractant protein-1 (MCP-1)19. Furthermore,
trans-fatty acids have been associated with increased levels of
several markers of endothelial activation, including soluble
intercellular adhesion molecule 1, soluble vascular-cell
adhesion molecule 1 and E-selectin20. Trans-fatty acids are
postulated to be involved in promoting vascular dysfunction,
as reflected by a reduction in brachial artery flow21. These
observations suggest that trans-fatty acids are linked to the
development of CHD, probably via a vascular pro-inflamma-
Although there is strong epidemiological evidence implicat-
ing elevated trans-fatty acid consumption in the development
of CHD, the extent and manner in which trans-fatty acids
affect the vasculature remain largely unknown. Clearly, vascu-
lar endothelial cells play a vital role in the development and
progression of atherogenesis. In the present study, we initiated
in vitro studies to determine the direct effects of trans-fatty
acid supplementation on the phenotypic and functional conse-
quences in HAEC. We hypothesised that trans-fatty acid
incorporation would induce a pro-inflammatory response lead-
ing to altered cell function.
Materials and methods
Chemicals and reagents were purchased from Sigma Chemi-
cal Company (St Louis, MO, USA), unless otherwise noted.
Becton Dickinson (Bedford, MA, USA). Consumable tissue
culture materials and Transwell inserts were acquired from
Fisher Scientific (Pittsburgh, PA, USA). Sphingosine-1-phos-
phate (SPP) was purchased from Calbiochem (La Jolla, CA,
USA). The protein growth factors utilised in the present
study and the MCP-1 ELISA kits were acquired from R &
D Systems, Inc. (Minneapolis, MN, USA). Human-derived
aortic endothelial cells as well as the EGM-2MV Bullet
kits (endothelial growth medium-2 microvascular) were pur-
chased from Cambrex (East Rutherford, NJ, USA). All
fatty acids were acquired from Nu-Chek Prep Incorporated
(Elysian, MN, USA).
Human aortic endothelial cell culture
A primary cell line derived from HAEC was maintained in
endothelial cell basal medium-2 (EBM-2) containing 5%
fetal bovine serum and the bullet kit materials as specified
by the manufacturer. Cells were maintained at 378C in a
humidified atmosphere in the presence of 5% CO2. Only
endothelial cell cultures of less than ten passages and
80–90% confluence were utilised in the present study.
Fatty acid incorporation into the endothelial cells
Stock solutions (1mM) of fatty acids (cis-18:2, linoleic acid;
trans-18:2, linoelaidic acid) were prepared by complexing
with fatty acid-free bovine serum albumin22. Sub-confluent
endothelial cells were cultured for 24h in EBM-2 complete
media either in the presence or absence of 25mM-cis-18:2
or -trans-18:2 fatty acid. This concentration of fatty acids
was found to be optimum by time- and dose-dependent assess-
ment of fatty acid effect on cell growth and morphology (data
not shown). After incubation, the cells were trypsinised and
repeatedly washed in PBS (Ca and Mg free) containing 1%
bovine serum albumin to ensure removal of NEFA. Lipids
were extracted with chloroform–methanol (2:1, v/v) using
the Folch method23. The lipid extracts were further fractio-
nated into phospholipids, TAG and cholesteryl esters by
TLC using a solvent system (hexane–diethyl ether–acetic
acid, 70:30:1, by vol.). The lipid fractions were scraped
from the TLC plate and subjected to acid-catalysed esterifica-
tion by heating at 1008C for 90min in a boron trifluoride–
methanol solution (14%). The methyl esters of fatty acids
were separated on a GC system (Shimadzu GC2010;
Shimadzu, Columbia, MD, USA) equipped with an Rt 2560
column (100m; 0·25mm internal diameter; 0·2mm). The
oven temperature was ramped from 1008C (4min hold) to
2408C at 38C/min (10min hold) with a flame ionisation detec-
tor at 2508C. Fatty acid peaks were identified by retention time
in comparison with authentic standards (Restek Corp., Belle-
fonte, PA, USA). Areas of identified peaks from 14:0 to
22:6n-3 were summed and individual fatty acids are
expressed as area percentage of total identified peak areas.
Data were analysed with Shimadzu’s GC solutions software
(Columbia, MD, USA).
Flow cytometric analysis of adhesion molecule expression
Trypsinised endothelial cells (1 £ 105/sample) were washed in
PBS containing 0·5% bovine serum albumin and re-suspended
into a volume of 100ml of this labelling buffer. Cells were
labelled with 0·25mg phycoerythrin-conjugated antibody for
20min; subsequently, the cells were washed twice in PBS con-
taining 0·5% bovine serum albumin. An isotype control was
established for each sample set to ensure specificity of the
antibody binding. Analysis was performed on a FACSCalibur
flow cytometer (Becton Dickinson, San Jose, CA, USA)
equipped with an air-cooled Ar laser emitting at a 488nm
wavelength. Fluorescence was detected through a 575 ^ 26
band pass filter and quantified using CellQuest Software
(Becton Dickinson). Results indicate the mean fluorescent
intensity of gated endothelial cells, which excluded cellular
debris and particles.
K. A. Harvey et al.724
British Journal of Nutrition
Endothelial cell adhesion to basement membrane components
HAEC (1 £ 104) cultured with fatty acids, as described above,
were placed onto fibronectin (5mg/cm2) or vitronectin (1mg/
cm2) coated twenty-four-well plates. Cells were incubated
for 30min at 378C. Aspirating cells from the wells terminated
the assay; subsequently, the remaining non-adherent cells
were removed by washing three times in PBS (Ca and Mg
free). The adherent cells were fixed in a 5% formaldehyde
solution. Adhesion was quantified by enumerating the
average number of cells observed within random fields of
view (200 £ ).
Leucocyte adhesion to endothelial cell monolayers
Leucocytes were isolated from normal human peripheral blood
in compliance with institutional guidelines. Neutrophil leuco-
cytes were selected by means of density gradient centrifu-
gation using the Ficoll–Hypaque technique as previously
described24. Monocytes were enriched on a Ficoll–Hypaque
gradient before a second density gradient centrifugation step
using a 1:1 isosmotic Percoll solution with PBS–citrate
(NaH2PO4, 1·49mM; Na2HPO4, 9·15mM; NaCl, 140mM;
C6H5Na3O7.2H2O, 13mM; pH 7·2) as previously described25.
The leucocytes were washed twice in Hanks balanced salt sol-
ution and re-suspended to a concentration of 1 £ 105cells/ml.
HAEC were grown with cis-18:2 or trans-18:2 fatty acids in
twenty-four-well tissue plates to near confluency before use.
Cells were washed to remove fatty acids, and neutrophils or
monocytes (1 £ 104) were loaded onto the endothelial cell
monolayers and maintained at 378C for 30min. Non-adherent
cells were aspirated from the wells. To ensure the removal of
remnant non-adherent cells, the monolayers were washed
three times with Hank’s balanced salt solution followed by fix-
ation of adherent cells in a 5% formaldehyde solution.
Adhesion to the endothelial cells was quantified by enumerat-
ing the average number of leucocytes observed within random
fields of view (200 £ ) by at least two blinded observers.
Samples were assayed in quadruplicate.
Monocyte chemoattractant protein-1 analysis by enzyme-
linked immunosorbent assay
MCP-1 released from endothelial cells into the culture media
was quantified using a Quantikine Human MCP-1 Immunoas-
say ELISA kit (R&D Systems, Minneapolis, MN, USA)
according to the manufacturer’s guidelines.
Endothelial cell migration assay
Endothelial cell migration was performed as previously
described26. Briefly, harvested HAEC were washed in
serum-free EBM-2 and re-suspended to a concentration of
1 £ 106cells/ml. Cells (1 £ 105) were placed onto an 8 mm
Transwell chamber and incubated for 30min at 378C to
permit anchoring to the filter. These inserts were placed into
wells containing 300ml serum-free EBM-2 containing SPP
to induce directed migration over a 4h incubation. To halt
the HAEC migration, cells were removed from the upper com-
partment and the migrated cells were fixed in a 5% formal-
dehyde solution. The cells were subsequently stained with
migrated cells. HAEC migration was quantified on a Leica
inverted fluorescent microscope (model no. DMI4000B;
Leica Microsystems, Wetzlar, Switzerland) by enumerating
the average cell number in three randomly selected fields of
view (200 £ ) on three separate filters27.
In vitro endothelial cell capillary morphogenesis assay
HAEC differentiation into capillary-like structures was
accomplished using a two-dimensional Matrigel-based assay
as previously described28. Briefly, cells (3·5 £ 104/well) treat-
ed with cis-18:2 or trans-18:2 fatty acids were placed into
Matrigel-coated twenty-four-well tissue culture plates. The
cells were incubated in the absence or presence of angiogenic
stimulants (hepatocyte growth factor or SPP) and maintained
for 16h at 378C in the presence of 5% CO2. Non-treated
control samples were maintained in serum-free EBM-2
media. Capillary-like structures were examined microscopi-
cally (40 £ ) using an inverted Olympus CK40 microscope
and random photomicrographs were taken. Quantification
of the capillary-like structures was accomplished by enumerat-
ing the number of multi-cellular nodes as previously
described28,29. Each sample was assayed in triplicate and
reproduced on at least three separate occasions.
Data are represented as mean values and standard deviations
of at least three determinants. Statistical significance between
datasets was determined using the Student’s t test. Overall
tests were performed using ANOVA. Pairwise comparisons
between groups were performed using Tukey’s multiple com-
parison test. When a calculated P value of less than 0·05 was
observed, statistical significance is indicated.
Fatty acid incorporation
Endothelial cells were cultured for 24h in EBM-2 complete
media either in the presence or absence of 25mM-cis-18:2
or -trans-18:2 fatty acids. Both cis- and trans-fatty acids
were incorporated in HAEC resulting in an increase in the
total PUFA fraction, a corresponding decrease in the MUFA
fraction and only a modest decrease in the SFA fraction
(Fig. 1 (A)). Data presented in Fig. 1 (B) indicate that trans-
18:2 fatty acids incorporated more efficiently than the
cis-fatty acid counterpart. The cellular content of trans-18:2
increased to 21·9 (SD 1·0) % of the fatty acid content in the
presence of 25mM-trans-18:2; however, a similar concen-
tration of cis-18:2 resulted in an increase to 13·2 (SD 1·6)
% of the cellular fatty acid content. We further analysed
fatty acid content in the phospholipid, TAG and cholesteryl
ester fractions of endothelial cells treated with cis-18:2 or
trans-18:2 fatty acids. Consistent with total cell homogenates,
more trans-18:2 fatty acids were enriched in phospholipids
than cis-18:2 fatty acids (cis-18:2 distribution: untreated,
1·9%; cis-18:2-treated, 22·5%; trans-18:2-treated, 1·2%.
Trans-18:2 distribution: untreated, 0%; cis-18:2-treated,
0%; trans-18:2-treated, 40·4%). However, a similar level
Trans-fatty acids and endothelial cells725
British Journal of Nutrition
of cis-18:2 and trans-18:2 enrichment was observed in the
TAG fraction (cis-18:2 distribution: untreated, 2·2%; cis-
18:2-treated, 16·7%; trans-18:2-treated, 2·1%. Trans-18:2
distribution: untreated, 0%; cis-18:2-treated, 0%; trans-
18:2-treated, 16·7%). No detectable amounts of cis-18:2 or
trans-18:2 were found in the cholesteryl ester fractions.
Adhesion molecule expression on endothelial cells
The relative expression of adhesion molecules, intercellular
(CD51/CD61) was determined in HAEC after cis-18:2 and
trans-18:2 treatment using flow cytometric analysis. Inter-
cellular adhesion molecule-1 surface expression level (mean
fluorescent intensity) was 32·8% higher in trans-18:2-treated
cells (154·0 (SD 10·9)) compared with that of cis-18:2-treated
cells (115·9 (SD 5·4)) (Table 1). Similarly, vitronectin receptor
(CD51/CD61) expression exhibited a 21·8% increase in the
trans-18:2 fatty acid-treated endothelial cells (95·6 (SD 4·2))
compared with that of cis-18:2-treated cells (78·5 (SD 6·9))
Human aortic endothelial cell adhesion to basement
After observing the increased expression levels of key endo-
thelial adhesion molecules, fatty acid-incorporated HAEC
were examined for their ability to bind preferentially to fibro-
nectin- and vitronectin-coated wells. Compared with cis-18:2,
trans-fatty acid-treated HAEC demonstrated a nearly 1·7-fold
increase in their ability to adhere to fibronectin (trans-18:2,
70·5 (SD 3·7) v. cis-18:2, 41·5 (SD 4·0); Table 1). Similarly,
an approximately 1·5-fold increase was observed in trans-
fatty acid-treated HAEC adherence to vitronectin (Table 1).
Leucocyte adhesion to human aortic endothelial cells
Increased adhesion molecule expression on endothelial cells is
often a key indicator of a pro-inflammatory state, which would
result in a greater capacity for leucocyte tethering and sub-
sequent binding and extravasation. We therefore isolated neu-
trophils and monocytes from normal peripheral blood to
determine the effect of cis-18:2 and trans-18:2 fatty acid
incorporation on leucocyte adherence to endothelial cell
monolayers. Both neutrophils (trans-18:2, 58·8 (SD 11·0) v.
cis-18:2, 20·0 (SD 7·0)) and monocytes (trans-18: 2, 61·3
(SD 12·5) v. cis-18:2, 21·3 (SD 7·5)) showed a nearly 3-fold
greater adherence to trans-18:2-treated HAEC than that
measured with cis-18:2-treated HAEC (Table 1).
Monocyte chemoattractant protein-1 release
In addition to the alterations of the cell membrane composition
leading to increased adhesion potential, increased cytokine pro-
vested 24h post-fatty acid incorporation. As shown in Fig. 2,
trans-18:2-treated endothelial cells (6·6 (SD 0·2) ng/106cells)
Fig. 1. Fatty acid composition of human aortic endothelial cells incorporated
with cis- and trans-18:2 fatty acids. Sub-confluent endothelial cells were cul-
tured for 24h in endothelial cell basal medium-2 complete media in the pres-
ence or absence of fatty acid (25mM). Incorporation of fatty acids was
analysed by GC (Shimadzu GC2010; Shimadzu, Columbia, MD, USA).
(A) Distribution of the fatty acid classes in treated endothelial cells: SFA (B),
MUFA ( ), PUFA (
). (B) Relative incorporation of cis- ( ) and trans-18:2
(B) fatty acids into endothelial cells. Results are expressed as percentage
composition. Data are means for at least three experiments, with standard
deviations represented by vertical bars. Data were analysed by using
ANOVA (P,0·001) and Tukey’s multiple comparison test. *Mean value
was significantly different from that of untreated endothelial cells (P,0·05).
†Mean value was significantly different from that of the cis-18:2-treated
cells (P,0·05). ‡ Non-detectable levels of trans-18:2.
Table 1. Fatty acid effects on endothelial cell inflammatory responses†
(Mean values and standard deviations)
Adhesion molecule expression (mean fluorescent intensity)
Adhesion to matrix components (cells/200 £ field of view)
Adhesion to inflammatory cells (cells/200 £ field of view)
ICAM-1, intercellular adhesion molecule-1.
*Mean value was significantly different from that of the cis-18:2-treated cells
†Assays were performed as described in the text. Data were analysed using
Student’s t test between groups (n 4).
K. A. Harvey et al.726
British Journal of Nutrition
released roughly two times more MCP-1 than cis-18:2-treated
HAEC (13·9 (SD 0·6) ng/106cells).
Sphingosine-1-phosphate-induced human aortic endothelial
The migratory potential of endothelial cells enriched with cis-
18:2 or trans-18:2 fatty acids in response to the bioactive
phospholipid SPP is presented in Fig. 3. As expected, SPP-
directed migration resulted in cell mobility through the
porous membrane under every condition. However, the
trans-18:2-treated HAEC (113·3 (SD 7·0) cells/field) were
35% more motile in response to SPP than those of cis-
18:2-treated (83·6 (SD 5·1) cells/field) endothelial cells.
Human aortic endothelial cells capillary morphogenesis
Endothelial cells stimulated with pro-angiogenic phospholi-
pids and/or protein growth factors characteristically develop
into a network of capillary-like structures on Matrigel matrix
supports. Fig. 4 (A) depicts a typical representation of the
capillary-like structures in response to either SPP or hepato-
cyte growth factor. The extent of this structural formation
was quantified and presented in Fig. 4 (B). Cis-18:2 fatty
acid-incorporated endothelial cells mimicked the robust capil-
lary morphogenic response of non-supplemented cells to both
SPP and hepatocyte growth factor. HAEC supplemented
with trans-18:2 fatty acids demonstrated an impaired
ability to form the capillary-like structures on Matrigel
supports and exhibited an 80% reduction in capillary
morphogenesis in the presence of SPP (trans-18:2, 8·3 (SD
0·6) v. cis-18:2, 42·7 (SD 5·5)) or hepatocyte growth factor
(trans-18:2, 3·7 (SD 1·2) v. cis-18:2, 22·3 (SD 4·9)). In
contrast, cis-18:2-treated cells’ ability to form capillaries
was not significantly different from that of untreated control
(SPP, 45·3 (SD 2·1); hepatocyte growth factor, 23·8 (SD 3·6)).
Endothelial cells are critical cellular components in the devel-
opment and progression of atherosclerosis. In response to
inflammatory stimuli, endothelial cells exhibit increased leu-
cocyte adherence, which can culminate in the development
of atherosclerotic plaques. In the present study, we set forth
to examine the ramifications of trans-fatty acid cellular incor-
poration on endothelial cell phenotypic and functional charac-
terisation. We found that the incorporation of trans-fatty acids
into endothelial cells enhanced the activation state of the cells
and leads to altered cell function.
During the present investigation we determined the effects
of trans-18:2 fatty acids, which have been shown to have a
positive association with CHD30, on endothelial cell function.
We found that approximately 2-fold greater levels of trans-
18:2 fatty acid were enriched in the phospholipids than that
of cis-18:2, whereas both trans-18:2 and cis-18:2 were
enriched to a similar extent in TAG fractions. This observation
suggests that trans-fatty acids were incorporated to a greater
extent in cellular membranes. However, the most surprising
observation was that the membrane phospholipid incorpor-
ation of trans-18:2 was nearly 40% of total fatty acids.
This level of enrichment appears to be excessive. It has
been demonstrated that fatty acids are efficiently incorporated
in the phospholipids of endothelial cells. In the freshly isolated
human umbilical endothelial cells, about 14% of the total fatty
Fig. 2. Effect of cis- and trans-18:2 fatty acids on monocyte chemoattractant
protein-1 (MCP-1) release. Endothelial cells (1 £ 106) were incubated with
cis- or trans-18:2 fatty acids for 24h. Subsequently, the cells were washed
and then incubated further with endothelial cell basal medium-2 media.
Supernatant fractions were harvested 24h post-fatty acid incorporation.
MCP-1 release was quantified using a Quantikine ELISA kit purchased from
R & D Systems (Minneapolis, MN, USA). Data are means for at least three
experiments, with standard deviations represented by vertical bars. The data
were analysed using ANOVA (P,0·001) and Tukey’s multiple comparison
test. *Mean value was significantly different from that of untreated endo-
thelial cells (P,0·05). †Mean value was significantly different from that of
the cis-18:2-treated cells (P,0·05).
Fig. 3. Effect of cis- and trans-18:2 fatty acids on sphingosine-1-phosphate-
induced endothelial cell chemotaxis. Endothelial cells (1 £ 105) treated with cis-
or trans-fatty acids for 24h were placed onto an 8 mm Transwell chamber
insert and incubated for 30min at 378C to permit anchoring to the filter. These
inserts were then placed into wells containing serum-free endothelial cell basal
medium-2 in the presence or absence of sphingosine-1-phosphate (SPP) for
4h. The migrated cells were fixed in a 5% formaldehyde solution and sub-
sequently stained with 40,6-diamidino-2-phenyindole (5mg/ml). Human aortic
endothelial cell migration was quantified on an inverted Leica fluorescent
microscope by enumeration in three randomly selected fields of view (200 £ )
and performed by at least two blinded individuals. (B), control; ( ), cis-18:2
(25mM); ( ), trans-18:2 (25mM). Data are means for at least three exper-
iments, with standard deviations represented by vertical bars. The data were
analysed using ANOVA (no treatment, P¼0·880; SPP-treated, P,0·001) and
Tukey’s multiple comparison test in SPP-treated cells. *Mean value was signifi-
cantly different from that of untreated endothelial cells (P,0·05). †Mean value
was significantly different from that of the cis-18:2-treated cells (P,0·05).
Trans-fatty acids and endothelial cells727
British Journal of Nutrition
acids in phospholipids are present as cis-18:1 fatty acid but
these levels greatly increased to 22% on culturing in the pre-
sence of fetal bovine serum31. Furthermore, in the human
endothelial cell line EA.Hy 926, when grown in the presence
of 100mM-cis-18:1 fatty acids, incorporation of cis-18:1 in
phospholipids was increased to 48% from a baseline
of 24%32. Interestingly, adipose tissues of patients with
peripheral artery disease contained about 27% of total fatty
acids as trans-fatty acids (21% trans-18:1 þ 6% trans-
18:2) and about 13% of total fatty acids were present as
trans-fatty acids (8% trans-18:1 þ 5% trans-C18:2) in the
atherosclerotic plaques33. The trans-fatty acids in human
erythrocyte membranes range from 1 to 2% for trans-18:1
and from 0·2 to 0·4% for trans-18:234,35. Total trans-fatty
acid levels (trans-18:1 þ trans-18:2) in adipocytes range
from 6 to 9%36. Another study demonstrated that levels of
total trans-fatty acids increased in the phospholipid fractions
of human serum from 1% to nearly 4% on a trans-fatty
acid-enriched diet after 4 weeks37. The phospholipid fraction
from the rat’s diaphragm showed accumulation of trans-
18:1 up to 5% after consuming a trans-fatty acid diet for 3
months. These observations indicate that levels of trans-
18:1 can be increased to a variable proportion in different tis-
sues on consuming trans-fatty acid diets. There are not enough
data available in the literature to compare C18:2 trans-fatty
acid enrichment in endothelial cells in animals or human sub-
jects on a diet rich in trans-fatty acids. It is clear from these
studies using trans-18:1 that endothelial cells can efficiently
incorporate long-chain PUFA. In the present study an exces-
sive enrichment of trans-18:2 in endothelial cells appears to
be unphysiological, but it remains to be seen if the extent of
this enrichment can be achieved in vivo or perhaps in a
human system. Furthermore, the present results indicated
that endothelial cells incorporated cis- and trans-18:2 fatty
acids at the expense of MUFA and SFA content. It is of inter-
est to note that trans-fatty acids, although unsaturated in
nature, structurally resemble SFA38. SFA typically occupy
the sn-1 position, whereas unsaturated fatty acids occupy the
sn-2 position in phospholipids. The present results demonstrat-
ing that trans-fatty acids are incorporated at the expense of
MUFA suggest that trans-fatty acids may be acylated on the
sn-2 position of phospholipids, imparting a more saturated
and hydrophobic character. Although the determination of
sn-1 v. sn-2 incorporation was beyond the scope of the present
investigation, more saturated phospholipids, especially those
containing trans-fatty acids, are known to attract cholesterol39.
This phenomenon plausibly alters cell membrane structure,
including redefining lipid raft and non-raft regions in size,
organisation and composition. Lipid rafts are important
for cellular signalling, as they provide docking sites for recep-
tors, co-receptors and mediators including adhesion mol-
ecules40. Our data also support this interpretation by
demonstrating that cell surface expression of adhesion mol-
ecules was greatly enhanced in cells grown in the presence
of trans-18:2 fatty acids.
HAEC adhesion molecule expression consistently corre-
sponded with endothelial cell adherence to basement mem-
brane components and leucocyte binding to the endothelium.
Enhanced adhesion molecule expression is often associated
with an inflammatory endothelial cell phenotype. Although
the elevated antigenic expression levels in trans-fatty acid-
treated HAEC were statistically significant, the increase was
modest in comparison with an acute cytokine-stimulated
response. However, the present study suggests that long-term
exposure of trans-fatty acids to the endothelium could result
in a gradual, cumulative chronic state of activation, which
could promote the development of atherosclerosis. Additional
evidence was found in the notable increase in the MCP-1
Fig. 4. Effect of cis- and trans-18:2 fatty acids on endothelial cell capillary
morphogenesis. Endothelial cells (1 £ 105) treated with cis- or trans-fatty
acids for 24h were placed onto Matrigel-coated wells as described in the
Methods section. Human aortic endothelial cells were then supplemented
with either sphingosine-1-phosphate (SPP; 500nM) or hepatocyte growth fac-
tor (HGF; 100ng/ml) and maintained for 16h at 378C in the presence of 5%
CO2. (A) Random photomicrographs (40 £ ) were captured to assess the
extent of the formation of the capillary-like structures. (B) The capillary mor-
phogenesis was quantified by enumerating the number of multicellular
nodes. (B), Control treatment; ( ), cis-18:2 (25mM) treatment; ( ), trans-
18:2 (25mM) treatment. Data are means for at least three experiments, with
standard deviations represented by vertical bars. The data were analysed
using ANOVA (no treatment, P¼0·017; SPP-treated, P,0·001; HGF-treated,
P,0·001) and Tukey’s multiple comparison test. *Mean value was
significantly different from that of untreated endothelial cells (P,0·05).
†Mean value was significantly different from that of the cis-18:2-treated
K. A. Harvey et al. 728
British Journal of Nutrition
released by trans-fatty acid-treated HAEC. This modest
increase in cytokine production, which attracts leucocytes to
the primed endothelium, could initiate a cellular infiltration
of macrophages, thereby initiating a cascade of plaque for-
mation and intimal thickening. Increased MCP-1 cytokine pro-
duction hasbeen correlated
Previous studies have demonstrated that SPP exerts pro-
angiogenic effects on endothelial cells, including increases
in barrier integrity, chemotaxis and capillary morphogen-
esis26,29,42,43. SPP-induced chemotaxis in endothelial cells
was further enhanced in the trans-fatty acid-treated HAEC.
Following migration to the site of wound healing, endothelial
cells differentiate into vessel linings, a process mimicked in
vitro by the assessment of capillary morphogenesis on Matri-
gel matrix supports29. The SPP-induced capillary-like struc-
tural formation was significantly impaired in the trans-fatty
acid-treated endothelial cells. This endothelial dysfunction
could translate into an inability of endothelial cells to repair
damaged vessel linings, complicating the pathogenesis of the
arterial damage. Furthermore, this process could explain
impairment in collateral growth that serves to compensate
for an arterial occlusion, especially in the coronary circulation.
Thus, trans-fatty acids may play an important role in the
development of CHD, and perhaps peripheral vascular disease,
through by inhibiting compensatory remodelling.
Endothelial cell apoptosis has been implicated in the pro-
gression of atherosclerosis, possibly even contributing to the
rupturing of atherosclerotic plaques44. A recent report by
Zapolska-Downar et al.45demonstrated that trans-fatty acids
induce endothelial cell apoptosis, which is consistent with
an effect of trans-fatty acids on the latter stages of plaque
development and/or subsequent rupturing of the plaques.
The induction of endothelial cell apoptosis observed by
Zapolska-Downar et al. required significantly higher trans-
fatty acid supplementation (up to 5mM). Using considerably
lower trans-fatty acid-treatments (25mM) under the same
24h time frame, we were unable to observe an increase in
early signs of apoptosis in HAEC using Annexin V–propi-
dium iodide staining techniques (data not shown). The trans-
fatty acid-induced alterations in endothelial cell activation
and function in the present study are clearly not due to the
initiation of apoptosis. These alterations implicate trans-fatty
acids in triggering the development of atherosclerosis and/or
accelerating the progression of the disease. Trans-fatty acids
may impart their effect by enhancing intrinsic signalling
mechanisms leading to a chronic, pro-inflammatory state.
In an investigation by Kummerow et al.46trans-fatty acid
incorporation into HAEC resulted in increased Ca influx in
combination with Mg depletion. Both linoelaidic (trans-
18:2) and elaidic (trans-C18:1) acids increased incorporation
of radiolabelled Ca intracellularly, whereas stearic (C18:0)
and oleic (cis-18:2) acids did not. The authors suggest that
this model is representative of endothelial cell calcification,
a hallmark characteristic of atherosclerosis, and that dietary
trans-fatty acids compound the effect of the relatively low-
Mg American diet on this process. While these modest
increases in Ca influx probably result from an alteration in
cell membrane fatty acid composition and properties, the
effect of free trans-fatty acids on endothelial cell function
were not included in their study.
Consumption of trans-fatty acids was correlated with
adverse affects on endothelial cell function in vivo19,47.
Increased plasma concentrations of biomarkers of inflam-
mation, including soluble intercellular adhesion molecule-1,
soluble vascular cell adhesion molecule-1 and E-selectin,
were associated with the trans-fatty acid content of the diet
in the Nurses’ Health Study, a cross-sectional investigation
of 730 CVD-free women20. The authors suggest that this
association could explain the significantly greater risk of
developing CVD based on the consumption of a high-trans-
fatty acid content diet. While soluble adhesion molecules
and inflammatory cytokines correlate with CVD in vivo20,48,
multiple factors could trigger such a response as a result of
the progression of the disease.
In conclusion, the present study provides evidence for a
direct effect of trans-18:2 incorporation on the activation
status and functional consequence of endothelial cells in
vitro, in the absence of stimulation factors found in
plasma. Consumption of diets high in trans-fatty acids
may induce long-term progressive changes in the endo-
thelium that could trigger the development of CVD. The
present study suggests minimising or eliminating the dietary
intake of trans-fatty acids might prevent the initiation of a
pro-inflammatory state leading to the subsequent develop-
ment of atherosclerosis. We realised that cis-18:2 and
trans-18:2 fatty acids were incorporated to a different
extent in endothelial cells when incubated with a similar
concentration (25mM) of these fatty acids, which was an
unexpected finding. It is possible that the altered biological
activities in trans-18:2-treated endothelial cells we observed
were simply due to a higher content of fatty acids and inde-
pendent of their geometric isomers. Further investigation
is required to study biological activities related to inflam-
mation in endothelial cells after incorporating similar
levels of fatty acids.
The authors wish to thank Mr Colin Terry for the statistical
analysis of the data and Dr Karen Spear for her editorial assist-
ance. The contract grant sponsor was Showalter Cardiovascu-
lar Fund (contract grant number S-2007-1).
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