Toll-Like Receptor 4 Mediates Endothelial Cell Activation Through NF-κB but Is Not Associated with Endothelial Dysfunction in Patients with Rheumatoid Arthritis
et al. (2014) Toll-Like Receptor 4 Mediates Endothelial Cell Activation Through NF-kB but Is Not
Associated with Endothelial Dysfunction in Patients with Rheumatoid Arthritis. PLoS ONE 9(6): e99053. doi:10.1371/journal.pone.0099053
Toll-Like Receptor 4 Mediates Endothelial Cell Activation Through NF-kB but Is Not Associated with Endothelial Dysfunction in Patients with Rheumatoid Arthritis
Rossella Menghini 0
Umberto Campia 0
Manfredi Tesauro 0
Arianna Marino 0
Valentina Rovella 0
Giuseppe Rodia 0
Francesca Schinzari 0
Barbara Tolusso 0
Nicola di Daniele 0
Massimo Federici 0
Angelo Zoli 0
Gianfranco Ferraccioli 0
Carmine Cardillo 0
Angelo Scuteri, INRCA, Italy
0 1 Department of System Medicine, University of Tor Vergata , Rome , Italy , 2 Division of Cardiology, MedStar Heart Institute , Washington, DC , United States of America, 3 Department of Internal Medicine, Catholic University Medical School , Rome , Italy , 4 Department of Rheumatology, Catholic University Medical School , Rome , Italy , 5 Center for Atherosclerosis, Policlinico Tor Vergata , Rome , Italy
Objective: To investigate the effects of TLR4 antagonism on human endothelial cells activation and cytokine expression, and whether the Asp299Gly TLR4 polymorphism is associated with better endothelial function in patients with rheumatoid arthritis (RA). Methods: Human aortic endothelial cells (HAECs) were treated with lipopolysaccharide (LPS), OxPAPC, and free fatty acids (FFA) at baseline and after incubation with the TLR4 antagonist eritoran (E5564). Cytokine expression was assessed by quantitative real-time PCR. In vivo endothelial function was assessed as brachial artery flow-mediated dilation (FMD) in RA patients with the wild type gene (aa) and with the Asp299Gly TLR4 polymorphic variant (ag). Results: In HAEC, TLR4 antagonism with eritoran inhibited LPS-induced mRNA expression of IL-6, IL-8, TNFa, CCL-2, VCAM and ICAM (P,0.05 for all) and inhibited Ox-PAPC-induced mRNA expression of IL-8 (P,0.05) and IL-6, albeit not to a statistically significant level (p = 0.07). In contrast, eritoran did not affect FFA-induced mRNA expression of IL-6 (P.0.05). In 30 patients with RA (15 with the ag allele) undergoing measurement of FMD, no differences in FMD and plasma levels of IL6, IL-8, VCAM, and ICAM were found between the aa and the ag phenotype (P.0.05 for all). Conclusions: TLR4 signaling in endothelial cells may be triggered by LPS and oxidized phospholipids, leading to endothelial activation and inflammation, which are inhibited by eritoran. Our in vivo investigation, however, does not support an association between the Asp299Gly TLR4 polymorphism and improved endothelium-dependent vasodilator function in patients with RA. Further study is needed to better understand the potential role of TLR4 on endothelial dysfunction in this and other patient populations.
Funding: This work as supported by an intramural grant of Tor Vergata University to Dr. Menghini. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Chronic inflammation represents a pivotal mechanism in the
pathogenesis of atherosclerosis . Interestingly, recent evidence
suggests that innate immunity may also contribute to the
development of vascular damage by interacting with inflammatory
pathways . In particular, toll-like receptors (TLRs) are
increasingly being recognized as a link between the innate
immune system, inflammation, and atherogenesis. This family of
innate immune receptors is expressed by endothelial cells, in which
they trigger various signaling pathways and lead to cell activation,
increased expression of inflammatory cytokines and adhesion
molecules, and endothelial dysfunction [3,4]. While initially
identified as sensors of microbial invasion, TLRs are now known
to be activated also by endogenous ligands produced in inflamed
tissues, potentially leading to further inflammation and
perpetuating an inflammatory milieu . Among them, TLR4, a receptor
for lipopolysaccharide (LPS) from Gram negative bacterial cell
walls, also exhibits affinity for fatty acids , extracellular matrix
components, fibrinogen, and various heat shock proteins . Of
note, TRL4 signaling leads to activation of NF-kB , a pathway
associated with andothelial injury , and TRL4 expression is
increased in human atherosclerotic plaques . Additionally, lack
of TLR4 reduces atherosclerosis and alters plaque phenotype in
apoE-deficient mice fed a high-cholesterol diet . In agreement
with these data, clinical evidence indicates that the Asp299Gly
TLR4 polymorphism, a functional variant in the TLR4 gene
(896ARG) that attenuates receptor signaling and diminishes the
inflammatory response to LPS , is associated with decreased
atherosclerotic risk . However, whether antagonism of TLR4
prevents TLR4-induced expression of inflammatory cytokines and
adhesion molecules in human macrovascular endothelial cells has
not been investigated in detail.
Rheumatoid arthritis (RA) is one of the most prevalent systemic
autoimmune diseases  and is associated with endothelial
dysfunction  and increased cardiovascular risk . This risk
is attributed to the presence of both traditional and non-traditional
risk factors, including inflammation and immunologic
abnormalities . A growing body of knowledge indicates that TLR4 may
play a relevant role of in the pathogenesis of autoimmune damage
in RA . In line with this evidence, the Asp299Gly TLR4
polymorphism is associated with decreased RA disease
susceptibility and lower baseline disease activity . However, whether
the presence of the Asp299Gly TLR4 polymorphism is associated
with better endothelial function compared with the wild type
genotype in patients with RA has not been studied.
The current investigations were therefore designed to test the
following hypotheses: 1) antagonism of TLR4 with eritoran
(E5564) inhibits the expression of inflammatory cytokines and
adhesion molecules in human endothelial cells; and 2) the presence
of the Asp299Gly TLR4 polymorphism is associated with better
endothelium-dependent vasodilation compared with the wild type
genotype in patients with RA.
Materials and Methods
In-Vitro Experiments: Cell Culture and Treatment
Human aortic endothelial cells (HAECs) were purchased from
Lonza (Basel, Switzerland) and cultured according to the
manufacturers instructions. All experiments were performed using
HAECs between the 2th and the 5th passage. HAECs were treated
with: LPS (Sigma Aldrich, St. Louis, MO) at a concentration of
100 ng/mL for 6 hours; ox-PAPC (oxidation products of
Hycult Biotech, Uden, The Netherlands), an antigenic epitope of
oxidized LDL, at a concentration of 100 mg/mL for 6 hours; or
long chain free fatty acids (FFA, oleic acid 500 mM+palmitic acid
500 mM) for 24 hours. Cells were incubated with 10 nM eritoran
for 30 minutes prior to treatments where indicated. Eritoran (Eisai
Inc., Woodcliff Lake, NJ) is a synthetic analog of lipid A and a
potent and specific antagonist of LPS action, which inhibits lipid A
binding to MD2 and terminates MD2/TLR4-mediated signaling
. At the end of treatments, HAECs where collected and used
for molecular analysis.
In-Vitro Experiments: Gene Expression Analysis
Total RNA was isolated from HAECs using Trizol reagents
(Invitrogen Corp, Eugene, OR). A total of 2 mg of RNA was
reverse-transcribed into complementary DNA (cDNA) using the
High Capacity cDNA Archive kit (Applied Biosystems, Foster
City, CA). Fifty nanograms of cDNA was amplified by real-time
polymerase chain reaction (RT-PCR) using an ABI PRISM 7500
System and TaqMan reagents (Applied Biosystems) and
normalized to 18S ribosomal RNA as an endogenous control. Each
reaction was performed in triplicate, and the relative gene copy
number was calculated as 22DDCt as previously described .
In-Vitro Experiments: Western Blots
Total protein was isolated from HAECs and western blots were
performed as previously described . The following antibodies
were used: anti-P65, anti-phosphoSer536 P65, anti-IkBa, and
Clinical Study: Study Population
Nonsmoker patients with a diagnosis of RA according to the
ACR revised criteria  and age- and sex-matched healthy
controls were enrolled in the study. None of the patients had
history or presence of hypertension, diabetes,
hypercholesterolemia, cardiovascular disease, vasculitis, or any other systemic
condition. All RA patients were on chronic treatment with either
disease modifying antirheumatic drugs (DMARDs), monoclonal
antibodies, or their combination. When used, aspirin, coxibs or
other nonsteroidal anti-inflammatory drugs were withdrawn at
least one week before the study; patients were allowed to use
acetaminophen (paracetamol) or tramadol as needed. The Disease
Activity Score 44 (DAS 44) was calculated for each patient. This
score assesses disease activity by including tender and swollen joint
count and the erythrocyte sedimentation rate. The level of disease
activity can be interpreted as low (DAS#2.4), moderate (2.4,
DAS#3.7), or high (DAS.3.7) . A DAS,1.6 corresponds
with being in remission according to the American Rheumatism
Association (ARA) criteria . The study protocol was conducted
according to the principles expressed in the Declaration of
Helsinki, was approved by the institutional Ethics Committee of
Tor Vergata University, and all participants gave written informed
Clinical Study: Laboratory Methods
EDTA-collected blood samples were drawn on the study day
after an overnight fast, immediately centrifuged for 15 min at
10006g and stored at 280uC until analysis.
Detection of autoantibodies. Rheumatoid factor (RF)-IgM
and RF-IgA (Orgentec Diagnostika GmbH, Mainz, Germany) and
anti-CCP antibodies (Axis Shield Diagnostics, Dundee, UK) were
measured using commercially available ELISA kits Equal volumes
for each sample were loaded according to each specific kits
instructions. The suggested cut-off levels were 20 U/mL for
RFIgM and RF-IgA and 5 U/mL for anti-CCP antibodies. Soluble
biomarkers: plasma levels of IL-6, IL-8, ICAM, VCAM and MCP-1
were measured using commercially available ELISA kits (R&D
Systems, Minneapolis, MN, USA). Equal volumes for each sample
were loaded according to each specific kits instructions.
Quantitative levels of cytokines were determined by comparison with
standard curves and reported as picograms or nanograms per mL
(pg/mL or ng/mL). The sensitivity of the test was of 0.7 pg/mL
for IL-6, 1.5 pg/mL for IL-8, 0.1 ng/mL for ICAM, 0.6 ng/mL
for VCAM and 5.0 pg/mL for MCP-1. Genomic DNA for TLR4
genotyping was prepared from frozen whole blood with the use of
a blood DNA isolation kit (Genomic Prep, Amersham Pharmacia
Biotech, Piscataway, NJ). Subsequent allele-specific PCR
amplification for the TLR4 allele Asp299Gly was performed according
to a previously described protocol .
Clinical Study: Endothelial Function Testing
Assessment of endothelial function was conducted in the fasting
state using a standardized ultrasound procedure . Brachial
artery reactivity, a test of endothelium-dependent vasodilation,
was assessed as previously reported . Briefly, participants lay
supine on a bed and were allowed to rest for at least 10 minutes.
During the rest period, participants were connected to a
continuous ECG monitor and a pressure cuff was applied around
the upper forearm. The left brachial artery was then visualized on
the anterior aspect of the arm, 215 cm proximal to the
antecubital fossa, using a Logiq E ultrasound machine (GE
Figure 1. Effects of eritoran on LPS-induced mRNA expression (arbitrary units) of TNFa (top left panel), CCL-2 (top right panel), IL-6
(middle left panel), IL-8 (middle right panel), VCAM (bottom left panel), and ICAM (bottom right panel). Values reported as mean6SD
(n = 5 per group). CT: control; ER: eritoran; LPS: lipopolysaccharide; ER/LPS: eritoran/lipopolysaccharide. *p,0.05 vs CT, ER, and LPS/ER.
Healthcare Italia, Milan, Italy) with a high-resolution probe
(12MHz linear array transducer). After baseline images and flow
measurements were obtained, the pressure cuff applied on the
forearm was inflated at 250 mmHg for 5 minutes. Blood flow was
measured during the first 15 seconds after cuff deflation, and
arterial image acquisition for diameter measurements was
performed between 60 and 90 seconds after cuff deflation. Arterial
diameter was measured from the anterior to the posterior interface
between the lumen and the endothelium at end diastole, incident
with the R wave on the ECG. Images were analyzed by an
investigator, different from the sonographer, blinded to image
sequence and clinical data of study participants.
For the clinical study, sample size calculation was based on
differences between the values of FMD in the three groups. Using
a 2-sided paired t test, a sample of 12 subjects in each group was
calculated to be necessary to detect a 2% difference in FMD with
80% power and a,0.05. With anticipation of up to 3 (i.e. 20%)
technically inadequate vascular studies, 15 participants per group
were enrolled to yield 12 evaluable participants in each group. All
group data are reported as mean 6 SD. Group differences were
analyzed by one way ANOVA, Fisher exact test, and unpaired
Student t test, as appropriate. All calculated p values are
twotailed, and a value of p,0.05 was considered to indicate statistical
significance. Statistical analyses were performed using
commercially available software.
In-Vitro Experiments: Eritoran Inhibits LPS-Induced mRNA
Expression of Inflammatory Cytokines in HAECs
To determine the effects of TLR4 receptor antagonism on
LPSinduced expression of inflammatory cytokines in HAECs, we
assessed mRNA levels of IL-6, IL-8, TNFa, and CCL-2 mRNA
after treatment with LPS, alone and following pretreatment with
eritoran for 30 minutes. Incubation with eritoran did not lead to
significant changes in mRNA levels of these cytokines compared to
control. Treatment with LPS alone for 6 hours induced a
significant increase in cytokine expression. In contrast, when
HAECs were treated with LPS following pretreatment with
eritoran, cytokine mRNA levels were similar to control (figure 1,
top and middle panels).
In-Vitro Experiments: Eritoran Inhibits LPS-Induced mRNA
Expression of Adhesion Molecules in HAECs
To assess whether TLR4 receptor antagonism impacts
LPSinduced expression of adhesion molecules in HAECs, we
measured mRNA levels of VCAM and ICAM after treatment
with LPS, alone and following pretreatment with eritoran for 30
minutes. compared with control, eritoran alone did not lead to
significant changes in VCAM and ICAM mRNA levels.
Treatment with LPS alone for 6 hours caused a significant increase in
mRNA levels of the adhesion molecules. In contrast, when HAEC
were treated with LPS following pretreatment with eritoran,
mRNA levels of VCAM and ICAM were similar to control
(figure 1, bottom panels).
In-Vitro Experiments: Eritoran Inhibits ox-PAPC-Induced
mRNA Expression of IL-8 in HAECs
To explore the effects of TLR4 receptor antagonism on
oxPAPC-induced expression of inflammatory cytokines in HAECs,
we assessed the effects of ox-PAPC treatment on mRNA levels of
IL-6 and IL-8, alone and following pretreatment with eritoran for
30 minutes. Eritoran did not affect cytokine mRNA levels
compared with control. ox-PAPC alone for 6 hours significantly
increased cytokine mRNA levels. In contrast, when ox-PAPC
treatment followed pretreatment with eritoran, mRNA levels of
IL-8 were significantly reduced when compared with ox-PAPC
alone (figure 2, top panel). Similarly, mRNA levels of IL-6 were
reduced; however, they failed to reach statistical significance
(p = 0.07) (figure 2, middle panel).
In-Vitro Experiments: Eritoran Does Not Affect
FFAInduced mRNA Expression of IL-6 in HAECs
To confirm that eritorans effects on inflammatory cytokines are
mediated by TLR4, we assessed the effects of FFA treatment on
mRNA levels of IL-6, alone and after incubation with eritoran for
30 minutes. As in the previous experiments, eritoran did not affect
IL-6 mRNA levels compared with control. FFA alone for 6 hours
led to significantly increased IL-6 mRNA levels. Consistent with
our hypothesis that eritorans effects on inflammatory cytokines
are mediated by TLR4, pre-treatment with eritoran did not affect
FFA-induced inflammatory cytokine expression (figure 2, bottom
In-Vitro Experiments: Eritoran Reduces LPS-Induced
Phosphorylation of NF-kB p65 Subunit and IkB-a in
To confirm that the effects of TLR4 antagonism on the
expression of inflammatory cytokines are mediated by a
modulation of the NF-kB pathways, we assessed phosphorylation of
NFkB p65 subunit and of IkB-a by western blotting (figure 3, top
panel). Compared with control, treatment with eritoran for 30
minutes did not induce phosphorylation of NF-kB p65 subunit
and of IkB-a. In contrast, treatment of HAECs with LPS for 6
hours caused an increase in the phosphorylation of NF-kB p65
subunit and IkB-a. Pretreatment with eritoran prevented the
LPSinduced phosphorylation of NF-kB p65 subunit (figure 3, bottom
left panel) and IkB-a (figure 3, bottom right panel).
Clinical Study: Study Population
Thirty patients and 15 age- and sex-matched healthy controls
took part in the study. Their baseline clinical characteristics are
reported in Table 1. The mean DAS 44 score of all the patients
was 2.2 (range: 0.53.9). The majority of the patients were treated
with a disease-modifying anti-rheumatic drug (27/30) and/or a
biologic agent (19/30). No significant differences were observed in
these parameters, disease activity, and drug treatment between the
two patient groups (all P.0.05).
Figure 3. Representative Western blots (top panel) and bar graphs showing the effect of eritoran on LPS-induced phosphorylation
of NF-kB p65 subunit (bottom left panel) and IkB-a (bottom right panel) (n = 5 per group). Bottom left panel: *p,0.05 vs CT; NS: p.0.05
vs LPS/ER. Bottom right panel: **p,0.005 vs CT; NS: p.0.05 vs LPS/ER.
Clinical Study: Brachial Artery Reactivity
Baseline brachial artery diameter was similar in the three groups
(3.760.6 mm, 3.360.5 mm, and 3.460.7 mm in the control, aa,
and ag group, respectively, p = 0.18). Flow-mediated dilation was
significantly higher in the controls compared to the RA groups
(p = 0.014). However, no significant differences in FMD were
observed between the two patient groups (p.0.05) (figure 4 and
figure 5, top left panel).
Clinical Study: Plasma Cytokines and Adhesion Molecules
in RA Patients
Plasma levels of IL-6 and IL-8, of MCP-1, and of VCAM and
ICAM were similar between the aa and ag groups (figure 5).
The main results of our in-vitro investigations are that, in
HAEC, TLR4 antagonism with eritoran: 1) inhibits LPS-induced
mRNA expression of the inflammatory cytokines IL-6, IL-8,
TNFa, and CCL-2, and of the adhesion molecules VCAM and
ICAM; 2) inhibits ox-PAPC-induced mRNA expression of IL-8
and of IL-6, albeit to a borderline significant level; and 3) reduces
LPS-induced phosphorylation of NF-kB p65 subunit and IkB-a.
These findings indicate that TLR4 induces activation of human
macrovascular endothelial cells through NF-kB-dependent
pathways, leading to the expression of genes regulating the production
of inflammatory mediators and adhesion molecules. Importantly,
TLR4-dependent activation of these pathways is not restricted to
exogenous ligands such as LPS, as mRNA expression of IL-6 and
IL-8 was also triggered by Ox-PAPC. These phospholipid
oxidation products are present in sites of chronic inflammation,
in apoptotic cell membranes, and in oxidized LDL , suggesting
a potential pathophysiologic role of Ox-PAPC-dependent LTR4
activation in atherosclerosis. Finally, as FFA-induced IL-6
expression was not affected by eritoran, our data suggest that
TLR4 activation in macrovascular endothelial cells is possibly
restricted to specific lipid ligands such as Ox-PAPC.
Our results are in agreement with the recent findings from Lu
and colleagues. These authors reported that, in HAEC and in
dermal microvascular endothelial cells, LPS induced an increase in
mRNA expression and synthesis of IL-6, as well as a more robust
gene expression of IL-8 and other inflammatory cytokines, ICAM,
VCAM, chemokines, growth factors, and adhesion molecules .
Our study shows that also Ox-PAPC, an endogenous byproduct of
phospholipid peroxidation, triggers TLR4-dependent expression
Table 1. Baseline Characteristics of Study Participants.
Participants per group (number)
Fasting blood glucose (mg/mL)
Disease Activity Score (DAS 44)
Anti-CCP antibodies level (U/mL)
Rheumatoid Factor IgM level (IU/mL)
Rheumatoid Factor IgA level (IU/mL)
DMARD: disease-modifying anti-rheumatic drug; anti-CCP: anti-cyclic citrullinated peptide antibodies; ESR: erythrocyte sedimentation rate. hsCRP: high sensitivity
Creactive protein; BMI: body mass index.
*p value calculated using Fisher exact test and #p value calculated using one way ANOVA; all other p values calculated using unpaired t test.
Values are reported as mean 6 SD unless specified otherwise.
Figure 4. Representative images of the baseline brachial ultrasound of a participant with the aa genotype (top left picture), of a
participant with the ag genotype (top right picture), and of the baseline and post-hyperemic ultrasound of a healthy control
(bottom pictures). The calculation of flow-mediated dilation (FMD) using the values collected in the healthy control is also reported.
Figure 5. Flow-mediated dilation (top left panel) and plasma levels of IL-6 (top right panel), IL-8 (middle left panel), VCAM (middle
right panel), ICAM (bottom left panel), and MCP-1 (bottom right panel) according to TLR4 genotype in study participants. Values
reported as mean6SD.
of inflammatory mediators, confirming and further expanding
previous observations . In addition, we also confirm previous
evidence that the synthesis of inflammatory mediators induced by
TLR4 signaling is mediated by activation of NF-kB , which has
been associated with endothelial injury . Our in vitro findings
complement the current understanding of the role of TLR4
signaling in the pathogenesis of endothelial dysfunction proposed
by Liang and colleagues.  In a series of elegant ex-vivo
experiments using vessels from type 2 diabetic mice with mutated
TLR4 (TLR42/2), these investigators demonstrated that TLR4
activation leads to the transcription of NADPH oxidase 1 and 4
and increased reactive oxidative species (ROS) generation. In turn,
the higher concentration of ROS impairs eNOS coupling, thereby
reducing nitric oxide (NO) production; blunts
endotheliumderived hyperpolarizing factor-mediated vasodilation; and
increases the synthesis of vasoconstrictor prostanoids by cyclooxygenase
1. Thus, activation of TLR4 in macrovascular endothelial cells
appears to trigger an inflammatory and proatherosclerotic
phenotype, with enhanced expression of cytokines and adhesion
molecules, increased ROS production, and abnormal synthesis of
vasoactive factors. Conversely, in our study, TRL4 blockade by
eritoran significantly inhibited LPS-stimulated expression of
inflammatory and adhesion molecules in macrovascular
endothelial cell cultures. In the same setting, TRL4 signaling inhibition
was also effective in blunting IL-8 responses to Ox-PAPC
treatment. These findings are in keeping with previous
observations of mitigated inflammatory responses to LPS in endothelial
cells pretreated by TRL4-blocking antibodies . Even more
importantly, our results are consistent with the recent evidence
that TLR4 antagonism may inhibit vascular inflammation and
atherogenesis in diabetic ApoE2/2 mice, supporting the concept
that TLR4 pathway is a potential therapeutic target .
The principal findings of our clinical study are that well-treated
RA patients, despite low disease activity, have impaired brachial
artery FMD and that the presence of the Asp299Gly TLR4
polymorphism is not associated with better endothelial function or
lower plasma levels of inflammatory cytokines, adhesion
molecules, and MCP-1. Of note, MCP-1 and its associated protein
MCPIP are increased in experimental models of RA and exert
detrimental effects on endothelial function by decreasing NO
bioavailability . Therefore, our investigation does not confirm
the hypothesis that the Asp299Gly polymorphism, which has been
reported to decrease TLR4 signaling activity , is associated
with better vasodilator function compared to the wild type in this
population. These results are apparently at odds with our and
other groups laboratory data that suggest LTR4-mediated
endothelial activation and dysfunction, and are not consistent
with the evidence that the Asp299Gly TLR4 polymorphism is
associated with a decreased risk of atherosclerosis . A number
of factors may account for our negative results. The impact of the
Asp299Gly variant on the endothelium may be modest and minor
differences in endothelial function may have been missed due to
small sample size and high variability of FMD. Alternatively, it is
possible that the effects of this polymorphism on endothelial
function are negligible in patients with RA, in whom other
inflammatory stimuli may represent the main players in the
pathogenesis of endothelial damage and atherosclerosis . In
this regard, it must be considered that patients with RA recruited
for our study were receiving optimal medical treatment and had
normal DAS 44 scores and low levels of inflammatory markers. It
might be postulated, therefore, that it was difficult under those
conditions to detect possible differences in endothelial activation
and vasodilator function related to polymorphisms of the TRL4
gene. However, our results are in line with other studies that did
not find a significant association between TLR4 Asp299Gly
polymorphism and risk for development and progression of
atherosclerosis, including a recently published meta-analysis [33
In conclusion, the results of our in vitro study indicate that
TLR4 signaling in endothelial cells may be triggered by both LPS
and endogenous ligands, such as oxidized phospholipids, leading
to endothelial cell activation and a proinflammatory phenotype.
Importantly, TLR4 activation by endogenous ligands is likely to
occur in the presence of a proatherosclerotic milieu, where it can
contribute to maintain and amplify endothelial activation and
vessel wall inflammation. Further investigations are needed to
better understand the potential impact of TLR4 polymorphisms
on endothelial function in this and other populations at onset of
the disease and while on high disease activity. Certainly the
findings of our in vivo investigation do not suggest that the
Asp299Gly TLR4 polymorphism is associated with improved
endothelial function in patients with RA, while treated and in low
Eritoran (E5564) was kindly provided by EISAI Inc., Woodcliff Lake, NJ.
Conceived and designed the experiments: RM MT VR MF AZ GF CC
NDD. Performed the experiments: RM AM VR GR FS BT. Analyzed the
data: RM UC MF CC. Wrote the paper: RM UC MT GF CC.
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