ABSTRACT
Title
Inflammation and Acute Coronary Syndromes: More Pieces to the Puzzle?
Authors
S. Costantino1, L.P. Ciuffreda1, R. Russo1, E. D’Aiuto2, P. Cirillo3, G. Petrillo3, G. Cimmino4, R. De Palma2, P. Golino4, F. Rossi1 and L. Berrino1.
1Department of Experimental Medicine, Pharmacology Division, Second University of Naples, Naples, Italy
2Department of Clinical and Experimental Medicine, Section of Clinical Immunology, Second University of Naples, Naples, Italy
3Department of Clinical Medicine, Cardiovascular and Immunological Sciences, Division of Cardiology, University of Naples “Federico II”, Naples, Italy
4Department of Cardiothoracic Sciences, Division of Cardiology, Second University of Naples, Naples, Italy
1Department of Experimental Medicine, Pharmacology Division, Second University of Naples, Naples, Italy
2Department of Clinical and Experimental Medicine, Section of Clinical Immunology, Second University of Naples, Naples, Italy
3Department of Clinical Medicine, Cardiovascular and Immunological Sciences, Division of Cardiology, University of Naples “Federico II”, Naples, Italy
4Department of Cardiothoracic Sciences, Division of Cardiology, Second University of Naples, Naples, Italy
Abstract
Acute coronary syndromes (ACS) generally occur following rupture of a coronary atherosclerotic plaque leading to intracoronary thrombus formation. We and othershave demonstrated that a local inflammatory reaction, i.e., at plaque level, characterized by lymphocytes/macrophages infiltration, is present in the culprit coronary lesion of patients with ACS.However, the complex functional interplay of the various T-cell subsets, leading to different cytokine/chemokine production in this disorder is far from being clear. Furthermore, little is known about the effect, at the gene expression level, of this cytokine/chemokine production.
Aim
Thus, the aims of the present study was to determine the pattern of cytokine/chemokine expression within the coronary circulation of patients with ACS and to clarify the effects of these cytokines on gene expression profile.
Methods
In a first series of experiments, blood samples were simultaneously obtained from the aorta (AO) and the coronary sinus (CS) during diagnostic coronary angiography in 44 patients with ACS (n=28) and stable angina (SA, n=16). Cytokine/chemokine expression profile was measured using Luminex technology, which allows measurement of up to 100 different analytes in the same 50 ul-samples. The following cytokines/chemokines were measured: RGF, Eotaxin, FGFβ, G-CSF, HGF, IGNα, IL10, IL12P40/P70, IL13, IL15, IL1β, IL1α, IL2, IL2R, IL4, IL5, IL6, IL7, INFγ, IP10, MCP1, MIG, MIP1α, TNFα, RANTES, VEGF. Intracoronary production of cytokines/chemokines was calculated as a Δ% of the CS/AO ratios.
In a second series of experiments, blood samples were obtained from the CS and the AO of 12 patients (ACS, n=8; SA, n=4) and used in experiment of gene induction. Endothelial cell lines were exposed to blood serum taken from the CS and thus to cytokines/chemokines produced in the coronary circulation. Following 6 hrs of incubation RNAs were extracted and used for microarray experiments. The microarrays were scanned using Agilent G2565AA Microarray Scanner System controlled byAgilent’s Scan Control Software version A 7.0.1 Chemiluminescent signals were quantified with Feature Extration 9.5.3 software.Expression values were analyzed and normalized by GeneSpring GX 11.5.using Student's t test (p < 0.01) with a Bonferroni multiple test correction. Of the significantly differentially expressed RNA, only those with greater than 2-fold increase or decrease were considered.Functional and network analyses of statistically significant gene were performed using Ingenuity Pathways Analysis 8.0 (IPA).
Result
In patients with ACS, a significant intracoronary production of IL1α (19.1±6.5 vs 0.1±5.4, p<0.05), Eotaxin (27.1±7.6 vs 3.2±6.9, p<0.05), RANTES (4.4±1.1 vs 3.1±2.7, p<0.05), IL6 (25.6±10.8 vs 4.0±3.2, p<0.05), INFγ (252±95 vs 28±32, p<0.01), IL1Rα (19.2±5.3 vs 3.1±4.5, p<0.05), TNFα(22.9±6.4 vs 5.3±8.1, p<0.01), IL13 (136±47 vs 22±19, p<0.01) was observed as compared to patients with SA. All other measured cytokines/chemokines did not differ significantly.
According with GeneSpring analysis, in patient with ACS we found 55 genes up-regulated and 88 down-regulated. Among the genes up-regulated, particularly interesting was the gene IL3RA (Fold Change= 2.6, p<0.01).
0The analysis with IPA showed that the up regulated genes were associated to pathways of chemokine signaling (CCL2, CCR3, FOS, MRAS), IL-3 signaling (IL3RA, JAK1, FOS, MRAS), IL-6 signaling (FOS, IL8, MRAS, NFKBIA, NKBIE) and Crosstalk between dendritic cells and NK cells (ACTC1, CD83, CD86, IL3RA, LTA).
Conclusions
In patients with ACS a significant intracoronary production of selected cytokines/chemokines occurs; this leads to the up-regulation of several genes involved in different inflammatory responses. We conclude that this disorder is characterized by a network of local inflammatory responses more complex than the well-characterized Th1/Th2 paradigm.
Acknowledgements
This work was supported by MIUR (PRIN) Grant.
Aim
Thus, the aims of the present study was to determine the pattern of cytokine/chemokine expression within the coronary circulation of patients with ACS and to clarify the effects of these cytokines on gene expression profile.
Methods
In a first series of experiments, blood samples were simultaneously obtained from the aorta (AO) and the coronary sinus (CS) during diagnostic coronary angiography in 44 patients with ACS (n=28) and stable angina (SA, n=16). Cytokine/chemokine expression profile was measured using Luminex technology, which allows measurement of up to 100 different analytes in the same 50 ul-samples. The following cytokines/chemokines were measured: RGF, Eotaxin, FGFβ, G-CSF, HGF, IGNα, IL10, IL12P40/P70, IL13, IL15, IL1β, IL1α, IL2, IL2R, IL4, IL5, IL6, IL7, INFγ, IP10, MCP1, MIG, MIP1α, TNFα, RANTES, VEGF. Intracoronary production of cytokines/chemokines was calculated as a Δ% of the CS/AO ratios.
In a second series of experiments, blood samples were obtained from the CS and the AO of 12 patients (ACS, n=8; SA, n=4) and used in experiment of gene induction. Endothelial cell lines were exposed to blood serum taken from the CS and thus to cytokines/chemokines produced in the coronary circulation. Following 6 hrs of incubation RNAs were extracted and used for microarray experiments. The microarrays were scanned using Agilent G2565AA Microarray Scanner System controlled byAgilent’s Scan Control Software version A 7.0.1 Chemiluminescent signals were quantified with Feature Extration 9.5.3 software.Expression values were analyzed and normalized by GeneSpring GX 11.5.using Student's t test (p < 0.01) with a Bonferroni multiple test correction. Of the significantly differentially expressed RNA, only those with greater than 2-fold increase or decrease were considered.Functional and network analyses of statistically significant gene were performed using Ingenuity Pathways Analysis 8.0 (IPA).
Result
In patients with ACS, a significant intracoronary production of IL1α (19.1±6.5 vs 0.1±5.4, p<0.05), Eotaxin (27.1±7.6 vs 3.2±6.9, p<0.05), RANTES (4.4±1.1 vs 3.1±2.7, p<0.05), IL6 (25.6±10.8 vs 4.0±3.2, p<0.05), INFγ (252±95 vs 28±32, p<0.01), IL1Rα (19.2±5.3 vs 3.1±4.5, p<0.05), TNFα(22.9±6.4 vs 5.3±8.1, p<0.01), IL13 (136±47 vs 22±19, p<0.01) was observed as compared to patients with SA. All other measured cytokines/chemokines did not differ significantly.
According with GeneSpring analysis, in patient with ACS we found 55 genes up-regulated and 88 down-regulated. Among the genes up-regulated, particularly interesting was the gene IL3RA (Fold Change= 2.6, p<0.01).
0The analysis with IPA showed that the up regulated genes were associated to pathways of chemokine signaling (CCL2, CCR3, FOS, MRAS), IL-3 signaling (IL3RA, JAK1, FOS, MRAS), IL-6 signaling (FOS, IL8, MRAS, NFKBIA, NKBIE) and Crosstalk between dendritic cells and NK cells (ACTC1, CD83, CD86, IL3RA, LTA).
Conclusions
In patients with ACS a significant intracoronary production of selected cytokines/chemokines occurs; this leads to the up-regulation of several genes involved in different inflammatory responses. We conclude that this disorder is characterized by a network of local inflammatory responses more complex than the well-characterized Th1/Th2 paradigm.
Acknowledgements
This work was supported by MIUR (PRIN) Grant.