Experimental Study Of Ability Of Nicotine To Induce Formation Of Aortic Aneurysms And Its Underlying Molecular Mechanism

Backgrounds: Smoking is a leading risk factor of the formation of aortic aneurysms. Because of the complex components in cigarette smoke, which component in it is the causative factor of aortic aneurysms has yet to be defined. In in-vitro experiments, nicotine can induce the expression of matrix metalloproteinases in vascular resident cells, so it may have a potential ability to induce the formation of aortic aneurysms. It has been also shown that JNK signal pathway was activated in aortic aneurysmal tissues from human and experimental animals, and α7-nAChRs, a receptor of nicotine, existed on the resident cells in aortic tissue. For this reason, we suppose that JNK signal pathway and α7-nAChRs may be involved in the formation of aortic aneurysms induced by nicotine.Methods: In-vivo experiments: To demonstrate whether nicotine have an ability to induce the formation of aortic aneurysms in ten-to-twelve-month-old male C57BL/6J mice, these mice were treated with nicotine, angiotension II or both for 28 days, with a control group in which the mice were perfused with saline. To obtain the evidence of JNK signal pathway involved in the formation of aortic aneurysms, SP600125, a JNK inhibitor, or DMSO was subcutaneously injected into the mice which simutaneously received the infusion of nicotine and angiotensin II for 28 days. In above experiments, the observational indexes include the incidence of aortic aneurysms, the mortality of the mice caused by the aortic rupture, the positive staining of matrix metalloproteinases and CD68 and their colocalization, as well as the p-JNK expression in aortic tissue. In-vitro experiments: To explore the mechanism underlying the expression of adhesion molecules and matrix metalloprotenases induced by nicotine in mouse macrophage line(RAW264.7), the cells were pretreated with SP600125( a JNK inhibitor) or PNU-282987(an α7-nAChRs blocker) before the supplement of nicotine, followed by an evaluation for the expression level of protein and mRNA of adhesion molecules and metrix metalloproteinases.Results: A combination of nicotine and lower dose of angiotensin II succeeded in inducing the formation of aortic aneurysms and early deaths in the older male C57BL/6J mice. In three treatment, the expression of matrix metalloproteinases was increased in the adventitia and media of aortic wall. Additionally macrophages were also recruited into the aortic adventitia. An interesting finding was the localized expression of metrix metalloproteinases in aortic media and the exclusive expression of either MMP-2 or MMP-9 in the area. Subsequently, in a separate experiment, a phenomenon was found that the formation of aortic aneurysms and deaths caused by aortic rupture in the mice infused with a combination of nicotine and lower dose of angiotensin II were suppressed by the JNK inhibitor. In-vitro experiment, nicotine induced the expression of p-JNK, VCAM-1, MMP-2 and MMP-9 in RAW264.7 cells in a dose-dependent manner, with an upregulating expression by 5ng/ml nicotine but a downregulating expreesion by 500ng/ml nicotine. However, the upregulation of expression of p-JNK, VCAM-1, MMP-2 and MMP-9 was significantly inhibited or abrogated by SP600125, PNU-282987. Moreover, the phosphorylation of JNK was quelled by PNU-282987.Conclusions: The combination of nicotine and angiotensin II have an ability to induce the formation of aortic aneurysms and early deaths caused by aortic rupture in the older male C57BL/6J mice. The localized and subtype-selective expression of matrix metalloproteinases in aortic media may play an important role in this process. The CD68-positive cells are participant in the formation of aortic aneurysms. The JNK signal pathway are involved in the formation of aortic aneurysms induced by nicotine and angiotensin II. In in-vitro experiments, it has been also demonstrated that RAW264.7 cells stimulated with nicotine express adhesion molecules and matrix metalloproteinases in a dose-dependent mannar, moreover α7-nAChRs-JNK signal axis regulates the expression of adhesion molecules and matrix metalloproteinases induced by nicotine in RAW264.7 cells.