| Abstract |
Atherosclerosis is a chronic inflammatory process of the vascular wall. It is mainly
preceded through interaction of monocytes with endothelial cells, and involves
chemokine signalling and adhesion molecule responses on both types of cells. Several
chemokine ligands and receptors have been extensively investigated and have been
proved to be involved in a more disease-specific way in atherosclerosis.
Chemokines are low molecular weight chemoattractant cytokines known to be major
regulatory proteins in leukocyte trafficking and activation. They consist of an expanding
family of approximately 50 ligands and 20 receptors which are classified into four subgroups.
Chemokines are involved in almost every inflammatory response and play an
important role in the pathogenesis of a wide variety of infectious and inflammatory
diseases, such as, atherosclerosis. Indeed, recent evidence suggests a pivotal role of
chemokines in important aspects of atherogenesis, thus rendering them potential
therapeutic target of coronary artery disease (CAD).
Fractalkine, the only member of the CX3C sub-family, has been implicated in
atherogenesis, since it can act both as a chemotactic agent recruiting monocytes and Tcells
in the site of inflammation, and as an adhesion molecule contributing to the
diapedesis of leukocytes in inflamed tissues. The receptor of fractalkine (CX3CR1) is a
seven transmembrane domain G protein-coupled receptor, and fractalkine binds to it with
high affinity, activating intracellular signalling and directly mediating monocyte
adhesion.
Through its unique structural and functional characteristics, fractalkine is a “perfect”
candidate for an exceptional role in the pathogenesis of atherosclerosis.
In the present study, we investigated the potential role of fractalkine in atherosclerosis in
two different settings. By applying a population genetic study we evaluated the impact of
functional polymorphisms in the CX3CR1 gene on susceptibility to coronary artery
disease. Then using an in vitro cell-culture model we evaluated the expression of
CX3CR1 in the presence or absence of inflammatory stimuli and after treatment with
agents with established anti-inflammatory properties.
Two single nucleotide polymorphisms have been described in the DNA sequence of the
CX3CR1 gene: V249I and T280M. The latter polymorphism has been associated with
reduced affinity of the receptor for its ligand. We evaluated the frequency of the above
two polymorphisms along with three common polymorphisms of chemokine ligands and
receptors (CCR2-64I, CCR5-Δ32, SDF1-3’A) in 210 angiographically diagnosed CAD
patients and 165 controls with normal coronary arteries. We constructed haplotypes and
combined genotypes. We concluded to a higher frequency of 280M containing genotypes
in homozygote or heterozygote state in the control group (24.3 vs. 33.9 p=0.04). We also
observed lower severity of CAD –expressed as mean number of diseased vessels- among
patients carrying the 280M allele (1.65 ±0.11 vs. 1.94 ±0.07 p=0.03). These results are in
agreement with previous observations implicating the presence of the 280M allele with
dysfunction of the FKN/CX3CR1 pathway, and indicate its role in the establishment and
progression of atherosclerosis.
We further evaluated differences in FKN/CX3CR1 activity in patients with CAD and
healthy controls. We measured, applying flow cytometry, expression of CX3CR1 in the
surface of peripheral blood mononuclear cells (PBMCs) isolated from 13 patients with
angiographically diagnosed CAD and 13 healthy controls. We observed higher rate of
CX3CR1-positive cells in the monocyte gate of patients compared to controls.
In order to further explore the possible implication of FKN/CX3CR1 in atherosclerosis
we evaluated the CX3CR1 behavior in the presence of inflammatory factors (TNF-α,
INF-γ, angiotensin I and angiotensin II) involved in the establishment of the
inflammatory micro-environment of the dysfunctional endothelium. Since, activated Tlymphocytes
and monocytes/macrophages are the PBMC-subpopulations most actively
implicated in atherosclerosis, the expression of CX3CR1 along with the expression of a
representative receptor of each major chemokine family (CCR5 and CXCR4) was
assessed by flow cytometry in two cell lines (THP-1 monocytes and Jurkat T-
lymphocytes) and primary monocytes isolated from healthy donors. Cells were evaluated
in naïve state and after treatment with TNF-α, INF-γ, ANG-I and ANG-II. We
demonstrated that INF-γ has the ability to significantly increase the CX3CR1 expression
of THP-1 monocytes. We did not observe any effect in the fluorescence intensity or the
rate of CX3CR1-positive cells in the rest of the studied cellular populations after
treatment with the above agents.
We further attempted to determine whether angiotensin converting enzyme (ACE)
inhibitors could decrease monocyte bioactivity through alteration of CX3CR1
expression. We treated THP-1 cells and primary monocytes with the ACE inhibitors,
captopril and lisinopril, and the angiotensin receptor blocker (ARB), losartan. Despite the
previously reported multiple anti-atherogenic properties of ACE inhibitors we concluded
that both captopril and lisinopril have no effect on THP-1 cells’ and primary monocytes’
chemokine-receptor surface profile. However, ARB losartan significantly increased the
rate and fluorescence intensity of CX3CR1-positive primary monocytes. This is a rather
unexpected effect since up-regulation of CX3CR1 could not be considered as an antiatherogenic
effect of ARBs. Nevertheless it is still an indication that angiotensin pathway
modulators have the ability to alter the chemokine receptor profile of monocytes.
In summary, we concluded that dysfunction of the CX3CR1/FKN pathway (mediated by
functional polymorphisms of the CX3CR1 gene) reduce susceptibility to CAD.
Furthermore, we demonstrated higher rate of CX3CR1-positive monocytes in CAD
patients compared to controls. The latter observation could be attributed to increased
levels of pre-inflammatory agents such as INF-γ that compose the inflammatory microenvironment
of the diseased vessel. The present data underline the role of the
FKN/CX3CR1 pathway in the inflammatory processes involved in atherosclerosis, thus
rendering it a potential therapeutic target.
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Μελέτη του ρόλου της FKN και του υποδοχέα της CX3CR1 στην δημιουργία αθηρωματικής βλάβης σε ασθενείς με στεφανιαία νόσο, συσχετισμός της νόσου με γενετικούς πολυμορφισμούς
