| Abstract |
Atherosclerosis is a complicated multivariate disease of medium and large arteries,
which leads to progressive accumulation of smooth muscle cells, fat and connective
tissue in the vascular intima and is also connected with a lot of environmental risk
factors interacting with the individuals genetic background.
Growing evidence is obtained on the inflammatory nature of atherosclerosis.
Several proinflammatory factors, chemoattractant cytokines (chemokines), binding
molecules play an important role in orchestrating the overall procedure. In modern
bibliography there is an increasing interest on the chemokines role in the
atherosclerosis, because of their involvement in important aspects of atherogenesis,
such as recruitment of inflammatory cells in vascular wall and proliferation of smooth
muscle cells in atherosclerotic plaques.
Interleukin 8 (IL-8), the standard chemokine of CXC subgroup appears to be involved
in vascular pathology. Based on literature data, specific polymorphisms of the
chemokine CXCL8 (IL-8) genes and its receptors are involved in the clinical
manifestation of other diseases such as bronchial asthma, multiple sclerosis and
RSV infection.
In this context two of the five known polymorphisms were examined, the -251 A / T
and 781 C / T of the promoter of the IL-8 gene and their frequency in relation to the
existence or absence of coronary disease.
For this purpose blood samples were collected from 300 CAD patients and
healthy controls. All the survey subjects were selected after undergoing
Percutaneous Coronary Intervention (PCI). The DNA samples were divided in
homozygous and heterozygous as for the studied polymorphism. Additionally there
was also confirmation of the existence and position of the polymorphism -251 A/T by
Sequencing.
In the process of genotypic-phenotypic correlation 241 patients and 157 controls
were included. In the same population was also recorded the clinical establishment of
coronary heart disease (stable-unstable angina, acute myocardial infarction). This
way it became possible the further division of the members of patients group in two
subgroups: a) Patients with Acute Coronary Syndrome (ACS group, n = 121) and b)
Patients with stable disease (non ACS group, n = 120). The analysis showed a
strong association between the two loci (D '= 0.93).
Also a predominance of homozygous was observed as for the 251A allele in the
group of the non-ACS patients compared with the group of ACS patients (OR = 0.49,
95% CI: 0.24-1.0, p = 0.07). A predominance of AA251TT781 combined genotype
was also observed to the group of non-ACS patients (OR = 0.43, 95% CI: 0.19-0.97,
p = 0.04).
The overall work leads to the conclusion that the common AA251TT781 genotype is
related with reduced risk of acute coronary syndrome in diagnosed coronary patients.
It was also revealed the involvement of IL-8 in the biochemical processes leading to
acute coronary syndromes.
Then we examined the correlation of the presence of these two polymorphisms (-
251A/T and 781C/T of the promoter of the gene IL-8) with the predisposition for
restenosis appearance after PCI. More specifically the prevalence of these SNP's
was tested in 201 patients with CAD who had undergone PCI and they had shown
signs of recurrent ischemia.
After angiographic re-examination, patients were divided into 2 subgroups according
to the presence or absence of restenosis ISR (≥ 50% reduction in vessel diameter
during retesting): 1) ISR group (n = 73) and 2) non-ISR group (n = 128). No
statistically significant differences in the frequency of two studied polymorphisms
were found between the two subgroups.
Nevertheless, a statistically significant increase of the frequency of the connected
TT251TT781 genotype was found in patients with restenosis, which suggests that the
genetic diversity of the IL-8 gene in some way affects the tendency to restenosis after
PCI.
Moreover, during the second part of the PhD, we studied the expression of the IL-8
and the receptors of it, in cultivation of human monocyte cell line (THP-1). The
cultivation and incubation of the cells proceeded in a nutrient medium consisting of
RPMI, HEPES, GLUTAMIN, FBS and antibiotics penicillin and streptomycin while
most incubation times refer to 24 hours. After the 24hours incubation and after
centrifugation of cultivations for taking the cell pellets, the supernatant liquid was
separated and cooled to -20 °C, in order to be used in measurement of IL-8
expression of the cells.
Initially, THP-1 cells were cultivated under the influence of inflammatory factors such
as lipopolysaccharide (LPS), AngI and AngII and the expression of the receptors
CXCR1 and CXCR2 in IL-8 was measured. The incubation lasted for 24 or 72 hours
and then the expression of the studied receptors was qualitatively and quantitatively
determined. The receptors were measured by flow cytometry (FACS) after their
labeling with fluorescent antibodies. What was observed during this procedure was
slight increase of the IL-8 receptors during the incubation (24 hours) of the cells with
LPS while AngI and AlgII didn’t seem to cause significant difference in their
expression. No significant difference was either observed after 72 hours incubation.
Thereafter, an attempt was made to suspend the action of LPS using medicines and
anti-inflammatory substances. Initially, Captopril, Lisinopril and Losartan were used.
These are 3 drugs used to treat CAD.
The first two (Captopril, Lisinopril) are inhibitors of angiotensin converting enzyme
(ACE), while Losartan is an antagonist of the receptor for AngII. There was then,
incubation of THP-1 cells with LPS and simultaneously with LPS and each of the
above three substances. The two inhibitors of angiotensin converting enzyme (ACE)
didn’t show any significant effect on the cells and the expression of the receptors. In
contrast, Losartan caused a consistent, repeatable, and significant dose-dependent
increase of the expression of the receptor CXCR1 and CXCR2 (less than the first
one) after 24 hours incubation. This result led to a need of further investigation for the
effect of Losartan and other medicines of the same class in the expression of the
studied receptors and of course of IL-8.
This time two new medicines of the same class (antagonists of the receptor for AngII)
were used for the same experiments, Telmisartan and Candesartan. Telmisartan
gave the similar results as Losartan while incubation in Candesartan didn’t seem to
influence the expression or the receptors.
Subsequently, in the cell cultivation the inflammatory substances AngI and AngII
were added in order to reveal their action in combination with the under study
medicines. Slight decrease in expression of receptors CXCR1 and CXCR2 during
incubation of cells with Angll and Losartan was observed, compared with their
expression after incubation only with Losartan or Telmisartan. The result did not
come up the same as with AngI or any other medical substance. This fact led to the
probability of AngII and Losartan action on a common receptor.
In addition, the expression of IL-8 was measured under anyone of the above
conditions, with the ELISA method.
There, the supernatants which were kept for this purpose were used. The
measurement of the IL-8 expression in supernatants of cultivated cells in
inflammatory conditions and in under medicine influence conditions, revealed
significantly increased expression of IL-8 under the influence of all three inflammatory
substances (LPS, AngI and AngII) and a corresponding reduction of these rates
during the attempt to inhibit the inflammation with the medicines used. Additionally
there showed a tendency towards inhibition of Lozartan’s action by the presence of
AngII. These experiments illustrate potential genetic and cellular mechanisms
through which an inflammatory substance (IL-8) can affect and be affected during the
complex process of atherosclerosis.
|
Search
