Influence of Interleukin 1β on the expression of bradykinin B1 and B2 receptor mRNA in vascular and cardiac cells

Abstract

Via activation of B1 and/or B2 receptors, kinins play important roles in mediating the pathogenesis of cardiovascular disease. After myocardial infarction, release of components of the kallikrein kinin system such as kallikrein, kiniogen as well as kinins is increased. Recent study from rat myocardial infarction (MI) model revealed that the known increase of bradykinin early after MI is accompanied by an upregulation of both bradykinin B1 and B2 receptor expression. Cytokines, such like IL1ß, which is also overexpressed during MI, are capable of modulating the release of bradykinin and kinin receptor expression in many cell systems under inflammatory conditions. To get a further view of the co-relationship between cytokine IL1ß and the expression of kinin B1 and B2 receptors in vascular and cardiac cell systems, the present study was carried out 1), to characterize the basic expression of B1 and B2 receptor in cardiac myocytes, cardiac fibroblasts and aortic smooth muscle cells, 2), to describe the possible influence of IL1ß on the expression of both kinin receptors in these cell types. If so, 3), to investigate the time pattern of this regulatory effects; and 4), using a myocardial infarction rat model to characterize whether a blocked IL1ß production by an inhibition of IL1ß converting enzyme inhibitor (ICEI) affects the release of kinin receptors in the heart. 2\. EXPERIMENTAL DESIGN in vitro: neonatal cardiac myocytes, fibroblasts and aortic vascular smooth muscle cells were cultured. After 12 h serum free medium treatment, total RNA was isolated. Using RNase protection assay (RPA) method, the amounts of B1R and B2R mRNA from these cell-RNA samples were investigated, basic expression of B1R and B2R mRNA was analysed for each cell system. To evaluate the influence of cytokine IL1ß on the expression of B1R and B2R mRNA in different cell types, cells were firstly treated with serum free medium, then, new media containing IL1ß was used. Cells were treated with IL1ß using different concentrations or for varying times. After treatment, total RNA was isolated from cells and the amounts of B1R and B2R mRNA were investigated. In vivo: Using a rat model, myocardial infarction was introduced by coronary artery ligation. Since the beginning of MI induction rats were treated with the IL1ß converting enzyme inhibitor HMR 3480. 3 weeks after coronary artery ligation, the hearts were excised and the left ventricles were separated. Total RNA was isolated from the infarcted ventricular myocardium, the amounts of B1R and B2R mRNA were analysed using RPA method. 3\. RESULTS It is shown that basic expression of bradykinin B1 and B2 receptors (B1R and B2R), could be detected in cultured cardiomyocytes and aortic vascular smooth muscle cells. In cultured cardiomyocytes, stimulation of cytokine IL1ß significantly upregulated both B1R and B2R mRNA expression. This effect was much more stronger on B1R (a factor of 25 fold) than on B2R (a factor of 3 fold). In aortic vascular smooth muscle cells, expression of both B1 and B2 receptor mRNA could be detected, however, IL1ß treatment did not influence their expression in the cells. In cardiac fibroblasts, expression of both B1 and B2 receptor mRNA was not detectable, and the addition of IL1ß could not induce their expression. In vivo studies showed that, after MI induction, administration of IL1ß converting enzyme inhibitor HMR 3480 downregulated the bradykinin B1 receptor expression in the infarcted area of the heart, but this treatment caused no significant changes in B2R expression. 4\. DISCUSSION Cardiomyocytes and aortic vascular smooth muscle cells constitutively express both bradykinin B1 and B2 receptors. Stimulation with the cytokine IL1ß significantly upregulated both receptor mRNA expression in cardiomyocytes but not in smooth muscle cells. In cardiac fibroblasts, expression of both kinin receptor mRNA could not be detected, and the addition of IL1ß failed to induce their expression. In vivo studies revealed that the administration of IL1ß converting enzyme inhibitor downregulated the bradykinin B1 receptor expression in the heart after MI induction. Based on these data, it can be concluded that the elevated release of cytokine IL1ß early after myocardial infarction may, at least in part, upregulate bradykinin B1 and B2 receptor expression through a cardiomyocyte-specific pathway.

Neueste Studien zeigen, dass der bekannte Anstieg von Bradykinin kurz nach dem Myokardinfarkt (MI) begleitet wird von einer Hochregulation der Expression der Bradykinin B1- (B1R) und B2-Rezeptoren (B2R). Zytokine wie Interleukin1ß (Il1ß) sind in der Lage die Bradykinin (BK)- und Kininrezeptorexpression zu beeinflussen. Diese Arbeit soll zeigen: 1\. Charakterisierung der basalen B1- und B2-Rezeptorexpression in kardialen Myozyten (CMC), kardialen Fibroblasten (CFB) und vasculären glatten Muskelzellen (SMC). 2\. Beschreibung des möglichen Einflusses von Il1ß auf die Expression beider Kininrezeptoren in diesen Zelltypen. 3\. Untersuchung welcher Zelltyp verantwortlich ist für diesen Effekt. 4\. Untersuchung ob Il1ß-Inhibition mit einem Interleukin Converting Enzym Inhibitor (ICEI) einen Effekt auf die Kininrezeptorausschüttung im Herzen hat. CMC, CFB und SMC aus neonatalen Ratten wurden mit und ohne Il1ß-Behandlung kultiviert. Aus diesen Zellen wurde die Gesamt-RNA isoliert und die Expression des B1R und B2R analysiert. Zusätzlich wurde bei Ratten durch die Ligation der RIVA ein MI ausgelöst. Mit der Induktion des MI begann die Behandlung mit dem ICE Inhibitors HMR3480. 3 Wochen später wurden die Gesamt-RNA aus den infarzierten Rattenventikeln isoliert und und auf die Expression von B1R und B2R untersucht. In CMC wird durch Stimulation mit Il1ß die B1R- und B2R mRNA Expression hochreguliert. Dieser Effekt war beim B1R (25fach) wesentlich stärker zu beobachten als beim B2R (3fach). In SMC konnte die B1R- und B2R mRNA Expression nachgewiesen werden, aber nicht durch Stimmulation mit Il1ß beeinflußt werden. In CFB konnte weder B1R- noch B2R mRNA Expression nachgewiesen werden. Diese konnte auch nicht durch Stimmulation mit Il1ß induziert werden. In vivo Studien zeigten, dass nach der MI Induktion und Gabe des ICEI HMR3480 die B1R mRNA Expression im infarzierten Gebiet des linken Ventrikels runterreguliert wurde, aber keinen Einfluß auf die B2R mRNA Expression hatte. Die erhöhte Ausschüttung des Cytokines Il1ß kurz nach dem MI könnte die B1R- und B2R mRNA Expression hochregulieren über eine CMC spezifischen Stoffwechselweg