Abdominal aortic aneurysms (AAA) represent a life-threatening condition characterized by diffuse inflammatory infiltration, extracellular matrix degradation, and dilatation of the aortic wall. While the specific factors initiating chronic inflammation in AAA remain unknown, enhanced vascular reactive oxygen species (ROS) production have been associated with chronic inflammation, atherosclerosis, and human abdominal aortic aneurysms[7
]. ROS are a collection of oxygen-derived molecules such as NO and superoxide. A number of specific enzymes can generate intracellular ROS, including xanthine oxidase (XO), NOS, and the NADPH oxidases. Our present studies demonstrate the causal roles of ROS produced by iNOS and NADPH oxidases in the oxidative stress and matrix degradation that lead to aneurysm formation. A deficiency in iNOS or inhibition of NADPH oxidase leads to decreased ROS production in the aortic tissues. Importantly, decreased ROS levels are directly correlated with inhibition of matrix destruction and aneurysm formation.
Inducible NOS is widely expressed in diverse cell types under transcriptional regulation by inflammatory mediators[16
] and has been implicated in the pathogenesis of many disorders including atherosclerosis, stroke, and arthritis[18
]. In the present study, we found that CaCl2
-induced mouse aneurysmal degeneration was associated with increased expression of iNOS mRNA in the aortic wall. We further tested the effect of iNOS in aneurysm formation by using iNOS knockout mice. Deficiency of iNOS partially attenuated aneurysm formation. This is in contrast to previous studies reported by Lee and colleagues[21
] that indicated that iNOS does not play a necessary role in the aneurysm pathology in the elastase-induced murine aneurysm model. This discrepancy is likely attributable to the use of different murine aneurysm models (ie, elastase-induced murine aneurysm model and CaCl2
-induced murine aneurysm model). The elastase-induced murine model of AAA relies on an initial pressure mediated mechanical injury followed by an inflammatory injury from infusion of pancreatic elastase. As such, this model may be more representative of the growth, rather than initiation, phase of an aneurysm. The CaCl2
-induced model depends on an inflammatory response to the local application of CaCl2
to the adventitial arterial surface. Thus, in this latter model, the expansion begins de novo from the inflammatory response. Both models involve a local arterial injury followed by inflammatory cell invasion, increased expression of MMPs, matrix destruction, and a corresponding increase in vessel diameter, recapitulating the key features of human aneurysms. Our findings are in agreement with studies done previously by Johanning et al showing[22
] that pharmacologic inhibition of iNOS limited expansion of elastase-induced aneurysm in rats.
Human AAA are associated with increased local activity of NADPH oxidases and XO[23
]. We examined the role of superoxide production by NADPH oxidase and XO in AAA formation using a specific inhibitor of NADPH oxidases, apocynin, and a specific inhibitor of XO, oxypurinol. We found that apocynin, but not oxypurinol, significantly inhibits mouse aneurysm expansion. These data suggest that NADPH oxidases are the major source of superoxide generation in the aortic tissue and play an important role in aneurysm formation. The profound inhibition of aneurysm formation by NADPH oxidase inhibition suggests a more important and direct role on aneurysm formation than iNOS where aortic expansion is limited to a lesser degree. The results are supported by the observation that SMC, resident mesenchymal cells, and macrophages, the major inflammatory infiltrates in aneurysmal aortic wall, express high levels of NADPH oxidases[25
ROS play an important role in cardiovascular homeostasis and inflammatory conditions. We further investigated NO production in the aorta. The concentrations of nitrite and nitrate (NOx), the stable end products of NO, were significantly elevated in CaCl2-treated aneurysmal tissues. As expected, this increase in NOx levels was inhibited by iNOS gene deletion. The data suggest that enhanced NO production in aneurysmal tissues is mainly catalyzed by iNOS. Furthermore, the aortic superoxide production by NADPH oxidases was measured. Superoxide production was not significantly inhibited by apocynin. It is important to point out that the absolute amount of superoxide production was significantly reduced in apocynin treated mice prior to normalizing with protein concentrations, because the aortas from untreated CaCl2-induced aneurysm mice were larger and had higher protein content than apocynin treated mice.
Several studies have shown that ROS can induce the expression of matrix-degrading enzymes such as MMP-2 and MMP-9[8
]. MMP-2 and MMP-9 degrade various extracellular matrix proteins and play an important role in AAA formation[11
]. We, therefore, investigated whether NADPH oxidase inhibitor, apocynin, XO inhibitor, oxypurinol, or iNOS deficiency inhibited the activities of MMP-2 and MMP-9 in the aortic tissues. As expected, CaCl2
-treatment markedly induced MMP-2 and MMP-9 activity in wild type mouse aorta. This increase in MMP activity was inhibited by apocynin treatment and iNOS gene deletion, but not oxypurinol treatment. The findings demonstrate one of the mechanisms through which ROS inhibition blocks aneurysm formation.
In summary, we report that iNOS deficiency and inhibition of NADPH oxidase activity protects aortas from AAA formation in a CaCl2-induced murine aneurysm model. These findings are consistent with the concept that oxidative stress plays a causal role in the pathogenesis of AAA formation. Furthermore, these studies suggest that early inhibition of iNOS-induced NO production and of superoxide production catalyzed by NADPH oxidases may be protective from the development of aortic aneurysm.