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Interleukin-8 (IL-8) or CXCL8 is a potent chemotactic factor that is involved in atherogenesis. IL-8 mediates its pre-inflammatory effects through interaction with CXCR1 and CXCR2. In the present study, we investigated the effects of angiotensin II (Ang II) on IL-8 synthesis and CXCR1/CXCR2 expression of THP-1 monocytes. IL-8 was measured in the culture medium using ELISA. Expression of chemokine receptors CXCR1 and CXCR2 was evaluated by flow cytometry. Results demonstrated that the addition of Ang II increased IL-8 production in the THP-1 monocytes. The Ang II type 1 receptor blocker (ARB) losartan significantly blocked Ang II-induced IL-8 production. Notably, losartan blocked LPS-induced IL-8 production by THP-1 monocytes and produced a small but statistically significant reduction of baseline IL-8 production of naïve THP-1 cells. Losartan also produced a statistically significant increase of fluorescence intensity of naïve CXCR1- and CXCR2-positive THP-1 monocytes, probably as a negative feedback effect secondary to IL-8 downregulation. In conclusion, we demonstrated that Ang II increased IL-8 production by THP-1 monocytes. Losartan significantly suppressed the latter effect, suggesting an AT-1 mediated pathway. Moreover, losartan suppressed the IL-8 production of naïve THP-1 monocytes and LPS-treated THP-1 monocytes, suggesting a broader spectrum of pleiotropic effects. Extrapolating this in vitro observation to in vivo pathways, we propose Ang II-induced IL-8 production by monocytes as another pre-atherogenic potential of Ang II that can be effectively blocked by the AT1 receptor blockade.

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Genetic polymorphy of the distal promoter region of the ST2 gene influences transcriptional activity and susceptibility to atopic dermatitis and asthma. Based on the inflammatory background of atherosclerosis we hypothesized that ST2 distal promoter genetic polymorphy could also affect susceptibility to coronary artery disease (CAD). To test our hypothesis we performed direct sequencing of a 825 bp locus of the distal promoter -with previously reported significant polymorphy in 63 angiographically diagnosed CAD patients and 63 age and sex matched controls with negative coronary angiography. We identified 13 polymorphisms spanning this region two of which (-27307 T/A and -27614 C/A) had allele frequencies greater than 0.05. We further genotyped 111 subjects by applying allele-specific real-time PCR for the -27307 T/A and 27614 C/A polymorphisms, thereby increasing our sample to 129 CAD patients and 108 age- and sex-matched controls. We identified no phenotype-genotype interactions between cases and controls. However, among case subjects the severity of CAD expressed as a mean number of diseased vessels was greater in -27307 A allele carriers and either allele carriers (-27614 A or -27307 A) than in non-carriers (2.56 ± 0.73 vs. 1.83 ± 0.84, adjusted P = 0.027; 2.47 ± 0.74 vs. 1.8 ± 0.83, adjusted P = 0.023). Additionally, either allele carriers (-27614 A or -27307 A) were significantly more common in the multi-vessel disease group (n = 54) than in the single-vessel disease group (n = 75). In conclusion, we reported two new polymorphisms in the distal promoter region of the ST2 gene that possibly influence susceptibility to severe CAD. The functional impact of these polymorphisms remains to be determined.

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Interleukin-8 is a strong mediator of inflammation and has been implicated in the biochemical pathways involved in a wide range of inflammatory diseases including atherosclerosis. We investigated the potential influence of two common functional polymorphisms of the interleukin (IL)-8 gene: -251A/T and 781C/T on susceptibility to in stent restenosis (ISR) following percutaneous coronary intervention (PCI). The hypothesis was tested by screening for the prevalence of the above polymorphisms in 201 coronary artery disease (CAD) patients subjected to PCI and presenting with symptoms or signs of recurrent ischemia. Patients were angiographically re-evaluated and formed the ISR group (n = 73) and the non-ISR group (n = 128) based on the presence or absence of ISR. One hundred and forty-seven subjects without angiographic evidence of CAD formed a reference control group (non-CAD group). A borderline statistically significant higher frequency of the TT(251)TT(781) combined genotype was observed in patients with ISR on re-evaluation compared with patients with normal follow-up angiography. The predominance of TT(251)TT(781) was independent of conventional risk factors for cardiovascular disease. Consequently, T(251)T(781) haplotype was significantly more common in the ISR group. The above observations indicate that the genetic diversity of the IL-8 gene influences patient susceptibility to ISR and suggests the implication of IL-8-mediated pathways in the process of ISR. However, the rarity of T(251)T(781) haplotype makes any clinical application of the above observations unfeasible.

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The potential regulatory effect of angiotensins on circulating mononuclear cell activation and migration has not yet been thoroughly evaluated. Using flow cytometry we assessed the possible effect of angiotensin I and II on the expression of CX3CR1 and a single representative of each major chemokine family (CCR5 and CXCR4) in THP-1 monocytes, Jurcat T lymphocytes and primary monocytes-isolated from healthy donors. Fluorescence intensity and the rate of chemokine-positive cells was measured in naïve cells and cells treated with angiotensin I and II. Neither angiotensin I nor angiotensin II exhibited any effect on fluorescence intensity and the rate of CX3CR1-, CCR5- and CXCR4-positive cells in primary peripheral blood mononuclear cells and Jurkat T cells. However, angiotensin II significantly increased the rate of CX3CR1-positive THP-1 cells. This effect was not attenuated by the pre-incubation of THP-1 cells with the AT-1 receptor blocker losartan, suggesting that this was not an AT-1-mediated effect. Angiotensin I and II had no effect on fluorescence intensity and the rate of CCR5- and CXCR4-positive THP-1 cells. In conclusion, angiotensin II increases the rate of CX3CR1-positive THP-1 cells. By extrapolating this in vitro observation to disease mechanisms, we speculate that angiotensin II induces up-regulation of CX3CR1 and promotes firm adhesion of circulation CX3CR1-positive monocytes on CX3CL1 expressing endothelial cells inducing vascular inflammation and atherogenesis.

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Since the establishment of the inflammatory basis of atherosclerosis, several pro- or anti-inflammatory agents have been examined as potential mediators of the biochemical pathways of lesion formation. Interleukin (IL)-8 was first characterized in 1987. Since then, knowledge regarding its role in leucocyte trafficking and activation has advanced rapidly, especially in the field of cardiovascular disease. In the scientific literature, there is sufficient evidence to support beyond any doubt the involvement of IL-8 in the establishment and preservation of the inflammatory micro-environment of the insulted vascular wall. However, how the information derived from in vitro studies and animal models can be applied in clinical practice has yet to be determined. In the present review, the available evidence regarding the role of IL-8 in cardiovascular disease is presented, and future perspectives are discussed.

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Regulated on activation, normal T cell expressed and secreted (RANTES) gene promoter is a regulatory region and a site of notable genetic diversity. In order to explore a possible interaction between RANTES promoter genetic diversity and susceptibility to coronary artery disease (CAD) and in stent restenosis (ISR), we initially sequenced a locus extending from -516 to 40 covering the entire region of the RANTES promoter in 100 subjects randomly selected from our cohort. Four single nucleotide polymorphisms (SNPs) were identified: -403G/A, -256G/A, -109C/T and -28C/G. The frequency of the -109C/T and -256G/A variations was <0.01, and was considered to be of limited significance. The frequency of the -403G/A and -28C/G polymorphisms was evaluated in the entire sample, which consisted of 118 patients subjected to percutaneous coronary intervention (PCI) without ISR on angiographic re-evaluation (no IRS group), 74 CAD patients with ISR on angiographic re-evaluation (IRS group) and 146 controls without angiographic evidence of CAD (no CAD group). No association was established between the RANTES promoter genotype and ISR. A genotype-phenotype interaction was observed between the -403G/A polymorphism and CAD. The -403A homozygotes were significantly more common in the CAD group than in the controls. The severity of CAD among case subjects, expressed as the mean number of diseased vessels, was significantly higher among -403A homozygotes as compared to wild-type homozygotes and heterozygotes. In conclusion, the RANTES -403A allele was associated with the presence and severity of CAD independently of conventional cardiovascular risk factors. The RANTES promoter genotype did not influence susceptibility to ISR in patients subjected to PCI.

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Interleukins (ILs) are key mediators in the chronic vascular inflammatory response underlying several aspects of cardiovascular disease. Due to their powerful pro-inflammatory potential, and the fact that they are highly expressed by almost all cell types actively implicated in atherosclerosis, members of the IL-1 cytokine family were the first to be investigated in the field of vessel wall inflammation. The IL-1 family is comprised of five proteins that share considerable sequence homology: IL-1alpha, IL-1beta, IL-1 receptor antagonist (IL-1Ra), IL-18 (also known as IFNgamma-inducing factor), and the newly discovered ligand of the ST2L receptor, IL-33. Expression of IL-1s and their receptors has been demonstrated in atheromatous tissue, and serum levels of IL-1-cytokines have been correlated with various aspects of cardiovascular disease and their outcome. In vitro studies have confirmed pro-atherogenic properties of IL-1alpha, IL-1beta and IL-18 such as, up-regulation of endothelial adhesion molecules, the activation of macrophages and smooth muscle cell proliferation. In contrast with this, IL-1Ra, a natural antagonist of IL-1, possesses anti-inflammatory properties, mainly through the endogenous inhibition of IL-1 signaling. IL-33 was identified as a functional ligand of the, till recently, orphan receptor, ST2L. IL-33/ST2L signaling has been reported as a mechanically activated, cardioprotective paracrine system triggered by myocardial overload. As the roles of individual members of the IL-1 family are being revealed, novel therapies aimed at the modulation of interleukin function in several aspects of cardiovascular disease, are being proposed. Several approaches have produced promising results. However, none of these approaches has yet been applied in clinical practice.

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INTRODUCTION: Interleukin 8 is a strong chemoattractant factor for neutrophils and T lymphocytes. We investigated the potential influence of two common polymorphisms of the interleukin-8 gene, -251A/T, and 781C/T on susceptibility to coronary artery disease. MATERIALS AND METHODS: The hypothesis was tested by screening for the prevalence of the above polymorphisms in 241 angiographically diagnosed coronary artery disease patients compared to 157 selected controls with negative coronary angiography. RESULTS AND DISCUSSION: We found no significant differences between cases and controls concerning the allelic and genotypic frequencies of both the studied polymorphisms. Nevertheless, a statistically significant lower frequency of the AA containing genotypes was observed in cases presenting with acute coronary syndromes compared to asymptomatic subjects or patients with stable coronary artery disease (OR = 0.44, 95%CI: 0.2-0.98, p = 0.04). The strongest statistical significance was observed in the AA(251)TT(781) combined genotype (OR = 0.34, 95%CI: 0.14-0.85, p = 0.02). CONCLUSION: Our results suggest that the genetic diversity of the interleukin-8 gene influences the clinical manifestation of CAD.

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