Ο ρόλος της χημοκίνης CXCL8 (IL-8) και των υποδοχέων της CXCR1 και CXCR2 στην παθογένεια της στεφανιαίας νόσου

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 189 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.