Immunosuppressive Drug Azathioprine Reduces Aneurysm Progression Through Inhibition of Rac1 and c-Jun-Terminal-N-Kinase in Endothelial Cells

Abstract

Objective— In aortic aneurysms the arterial vessel wall is dilated because of destruction of its integrity, which may lead to lethal vessel rupture. Chronic infiltration of inflammatory cells into the vessel wall is fundamental to aneurysm pathology. We aim to limit aneurysm growth by inhibition of inflammation and reducing endothelial cell (EC) activation with immunosuppressive drug azathioprine (Aza). Approach and Results— Aza and its metabolite 6-mercaptopurine have anti-inflammatory effects on leukocytes. We here demonstrate that treatment of ECs with 6-mercaptopurine inhibits cell activation as illustrated by reduced expression of interleukin-12, CCL5, CCL2, and vascular cell adhesion molecule-1 and inhibition of monocyte–EC adhesion. The underlying mechanism of 6-mercaptopurine involves suppression of GTPase Rac1 activation, resulting in reduced phosphorylation of c-Jun-terminal-N-kinase and c-Jun. Subsequently, the effect of Aza was investigated in aneurysm formation in the angiotensin II aneurysm mouse model in apolipoprotein E–deficient mice. We demonstrated that Aza decreases de novo aortic aneurysm formation from an average aneurysm severity score of 2.1 (control group) to 0.6 (Aza group), and that Aza effectively delays aorta pathology in a progression experiment, resulting in a reduced severity score from 2.8 to 1.7 in Aza-treated mice. In line with the in vitro observations, Aza-treated mice showed less c-Jun-terminal-N-kinase activation in ECs and reduced leukocyte influx in the aortic wall. Conclusions— The immunosuppressive drug Aza has an anti-inflammatory effect and in ECs inhibits Rac1 and c-Jun-terminal-N-kinase activation, which may explain the protective effect of Aza in aneurysm development and, most importantly for clinical implications, aneurysm severity.

Inhibition of GTPase Rac1 in Endothelium by 6-Mercaptopurine Results in Immunosuppression in Nonimmune Cells: New Target for an Old Drug

“…Azathioprine, 6-MP and the downstream metabolite 6-T-GTP have been shown to decrease Rac1 activation in T cells (11), which could be the underlying mechanism in endothelial cells as well. Indeed, we recently revealed that TNF-a-induced Rac1 activation is impaired when endothelial cells are pretreated with 6-MP, by pulldown of active Rac1 (19). In this study, we show that pretreatment of the endothelial cells with 6-T-GTP also blocked TNF-a-induced Rac1 activation to a similar extent as 6-MP and Rac1 inhibitor ITX-3, in an ELISA-like setup (Fig.…”

“…To accomplish leukocyte adhesion, cell surface adhesion molecules are essential. Previously, we have demonstrated in TNF-a-stimulated endothelial cells that 6-MP reduces the mRNA expression level of VCAM-1 but not of ICAM-1 (19). In this study, we show that 6-MP also suppresses VCAM-1 protein expression.…”

“…In TNF-a-stimulated endothelial cells that were pretreated with 6-MP, IL-6, IL-8, and IP-10 are significantly downregulated (Fig. 3) in addition to IL-12, CCL2, and CCL5 that we reported earlier (19). The fact that IL-1b mRNA expression is not suppressed shows that 6-MP inhibits specific proinflammatory pathways.…”

“…We have recently shown that low-dose azathioprine effectively prevents the chronic inflammatory aortic disease of abdominal aortic aneurysm formation in mice, by reducing endothelial JNK activation and macrophage infiltration of the vessel wall (19). To date, there is limited information on the exact cellular mechanism underling the immunosuppressive properties of azathioprine in T cells (10,11) and monocytes/macrophages (33).…”

“…Previously, we have shown that 6-MP can potently reduce the mRNA expression level of proinflammatory cytokine IL-12 and chemokines CCL2 and CCL5, also known as MCP-1 and RANTES, respectively (19). In this study, more cytokines and chemokines are studied, namely IL1b, IL-6, IL-8, as well as CXCL10, also known as IFN-g-induced protein 10 (IP-10).…”