Angiotensin II increases urokinase-type plasminogen activator expression and induces aneurysm in the abdominal aorta of apolipoprotein E-deficient mice

Abstract

Urokinase-type plasminogen activator (uPA) is increased in human abdominal aortic aneurysm (AAA). Chronic infusion of angiotensin II (Ang II) results in AAA in apolipoprotein E-deficient mice. We tested the hypothesis that Ang II infusion results in an elevation of uPA expression contributing to aneurysm formation. Ang II or vehicle was infused by osmotic pumps into apoE-KO mice. All mice treated with Ang II developed a localized expansion of the suprarenal aorta (75% increase in outer diameter), accompanied by an elevation of blood pressure (22 mmHg), compared to the vehicle-treated group. Histological examination of the dilated aortic segment revealed similarities to human AAA including focal elastin fragmentation, macrophage infiltration, and intravascular hemorrhage. Ang II treatment resulted in a 13-fold increase in the expression of uPA mRNA in the AAA segment in contrast to a twofold increase in the atherosclerotic aortic arch. Increased uPA protein was detected in the abdominal aorta as early as 10 days after Ang II infusion before significant aorta expansion. Thus, Ang II infusion results in macrophage infiltration, increased uPA activity, and aneurysm formation in the abdominal aorta of apoE-KO mice. These data are consistent with a causal role for uPA in the pathogenesis of AAA.

Figure 1.

Representative pictures show that localized expansion of the abdominal aorta between the diaphragm and the renal artery was found only in mouse infused with Ang II for 1 month (top) but not in control animal infused with PBS (bottom).

Figure 2.

Ultrasound images obtained noninvasively from a live apoE-KO mouse treated with Ang II for 1 month. Top: Longitudinal view of the suprarenal aorta including both the aneurysmal and adjacent nonaneurysmal segment. Bottom left: Cross-sectional view of the adjacent nonaneurysmal segment of the suprarenal aorta with an inner diameter of 0.88 mm. Bottom right: Cross-sectional view of the aneurysmal portion of the suprarenal aorta with an inner diameter of 2.26 mm.

Figure 3.

Direct measurement of the cross-section of the suprarenal aorta in apoE-KO mice treated with Ang II or vehicle for 1 month. Left: Representative sections of the suprarenal aorta from a mouse treated with Ang II (top) and one with vehicle (bottom). Right: Quantification of the outer and luminal diameters, and wall thickness of suprarenal aortas in apoE-KO mice treated with Ang II or vehicle. *, P < 0.05 between two groups.

Figure 4.

A: Day 10 lesion. Inflammatory cell infiltration of the adventitia is prominent. There is also an abrupt, focal, full-thickness necrosis of the arterial wall (arrow). This lesion appears as an acellular, pale, eosinophilic area. B: Day 20 lesion, again there is an abrupt lesion involving destruction of the vascular media. Note that activated monocytes in tissue are infiltrating into the lesion from the adventitial side (arrowheads). An area of acute hemorrhage is seen at asterisk. H&E stain (both A and B). C: Small atheroma containing foamy macrophages. Note that there is destruction of the top layer of the elastic laminae directly below the lesion. D: Extensive fibrosis in the adventitia. These changes are seen around areas where there has been destruction of the vascular media as well as beneath areas with intact elastic laminae. Note that in both C and D smooth muscle cells in the media are hypertrophied (elastin van Gieson stain, tissues from day 30).

Figure 5.

Ex vivo IL-6 secretion in the atherosclerotic aortic arch, and suprarenal and infrarenal aortas in apoE-KO mice infused with Ang II or vehicle for 1 month.

Figure 6.

Expression of uPA, PAI-1, and tPA in aneurysm. Total RNA extracted from pooled aortic tissues from 10 mice was subjected to quantitative RT-PCR using primers and probes specific for uPA (A), PAI-1 (B), and tPA (C).

Figure 7.

Expression of uPA and MMP protein in aneurysm. A: Immunoblotting to detect uPA protein in the aneurysmal (left) and nonaneurysmal (right) portion of the abdominal aorta in the mice treated with Ang II for 10 or 20 days. Two-chain uPA is detected as a 50-kd band. The recombinant mouse uPA (rm-uPA) B chain (5 μg) appears as a 30-kd band. B: Zymographic analysis of uPA activity. Extract from aneurysm tissues (AAA) (containing 10 μg of protein) was compared with a kidney extract from a wild-type mouse containing abundant uPA and with rm-uPA (0.1 μg). C: Gelatin zymography of tissue extracts of aneurysm and aortic arch from Ang II-infused mice. The pro- and activated forms of MMP-9 and MMP-2 were detected.