The major finding of the present study is that CyPA is a novel mediator of abdominal aortic aneurysm (AAA) formation. We characterized four pathologic mechanisms by which vascular CyPA promotes AAA formation (Supplement Fig. 7
). First, AngII-induced ROS promoted secretion of CyPA and proMMP-2. Second, secreted extracellular CyPA contributed to ROS production synergistically with AngII in VSMC. Third, CyPA promoted activation of MMP-2, by inducing MT1-MMP and augmenting ROS generation. Fourth, CyPA stimulated recruitment of CD45+
inflammatory cells. The source of CyPA responsible for AAA formation appeared to be cells resident in the vessel wall, especially VSMC since no AAA were observed in Apoe−/−Ppia−/−
mice after transplantation with Ppia+/+
bone marrow cells. Also, overexpression of CyPA in VSMC enhanced vascular ROS production, MMP activation, and AAA formation. Therefore, we propose a key role for vascular CyPA in AAA formation and other cardiovascular diseases associated with inflammation.
Daugherty’s group characterized the temporal events in AngII-induced AAA formation24
. First, they described medial accumulation of macrophages in regions of elastin degradation. Second, medial dissection occurred with dilation of the lumen and thrombus formation. Third, an inflammatory response comprised of macrophages and T and B lymphocytes was observed. Fourth, a repair response including elastin fiber regeneration and reendothelialization occurred; and finally there was neovascularization of the thrombus and vascular wall.
VSMC appear to be essential for AngII-induced AAA formation. Expression of the AT1a receptor, responsible for CyPA secretion, ROS generation and MMP activity, is highest in VSMC. In situ measurements of ROS generation and MMP activity were greatest in medial cells that stained for αSMA. Cultured VSMC from transgenic mice and human AAA lesions recapitulated the findings of increased ROS and MMP activation. Finally, bone marrow transplantation showed a minor role for hematopoietic cells. Specifically, our data suggest that VSMC-derived CyPA initiated AAA formation by promoting accumulation of macrophages. Apoe−/−Ppia−/− mice had significantly attenuated vascular ROS production, MMP activation, and MCP-1 secretion resulting in decreased macrophage accumulation. Overexpression of CyPA in VSMC enhanced ROS production and MMP activation, and caused AAA formation even in Apoe+/+ mice (). Finally, transplantation of bone marrow cells from Ppia+/+ mice into Apoe−/−Ppia−/− mice did not induce AAA formation, indicating that cells resident in the vessel wall were essential for AAA formation.
Our novel data show that extracellular CyPA induces ROS production in VSMC, which is consistent with our previous report that extracellular CyPA stimulates at least 3 signaling pathways (ERK1/2, Akt and JAK) in VSMC19
. These signaling pathways have been shown to be important for ROS production2,3
. Furthermore, ROS stimulate secretion of CyPA from VSMC19,21
. These reports and the present data suggest that CyPA plays a crucial role in VSMC through ROS generation. AngII is thought to induce the generation of ROS and thereby activates MMPs26
, thus leading to the onset of vascular inflammatory cell migration and AAA formation7,16,24
In the present study, CyPA deficiency reduced secretion of proMMP-2 and MMP-2 as well as MT1-MMP expression, all of which can be explained by reduced ROS production. Additionally, AngII has been shown to generate ROS and activate MMP-2 in a p47phox-dependent manner in the same model7,15,35
. VSMC-derived MMP-2 promotes degradation of collagen and elastin, contributing to the AAA formation29,30
. Expression of MT1-MMP is important for activation of MMP-2 in AngII-induced AAA formation36
. Besides enzymatic cleavage and activation of MMP-2 by cell surface expressed MT1-MMP30
, ROS have been shown to directly activate MMP-232
. AAA formation results from the synergistic activation of ROS production, MT1-MMP, and MMP-2. Therefore, CyPA appears to be a central mediator of AngII-mediated AAA formation.
The identification of CyPA as a mediator of tissue damage associated with inflammation and oxidative stress provides insight into the mechanisms of several therapies. For example, the Rho-kinase inhibitor Y27632, and simvastatin significantly reduced CyPA secretion from VSMC. Rho-kinase is an important therapeutic target in cardiovascular disease37
and Rho-kinase inhibition has been reported to reduce AngII-induced AAA formation38
. AT1a receptor blockers and ACE inhibitors have been shown to prevent AAA formation in mice39–41
. Based on the present study, reduced CyPA secretion may partially contribute to the therapeutic effect of these drugs on AAA formation. Because inflammation and oxidative stress contribute to tissue damage in several situations such as ischemia-reperfusion injury in the brain, heart and kidney, future studies of CyPA-mediated function in appropriate models may reveal a significant role in other diseases.
EMMPRIN, a putative CyPA receptor, was identified as a tumor cell membrane protein that is expressed in VSMC, activated by ROS and stimulates MMP production42
. A recent paper demonstrated ROS-dependent increases in EMMPRIN43
, which may be activated by binding of extracellular CyPA31
. Moreover, it has been demonstrated that EMMPRIN is strongly expressed in human AAA lesions44
. Therefore, it is logical to propose that agents which prevent CyPA binding to its receptors may have therapeutic potential. In summary, these reports and the present study suggest that extracellular CyPA and its receptor(s) represent novel therapeutic targets, particularly for AAA progression.